Clam shell blood reservoir holder with index line

ABSTRACT

A rotatable rotor for separating components contained in a fluid, comprising an annular base having a first annular channel and an annular cover having a second annular channel for holding a flexible doughnut-shaped centrifuge bag therebetween. The cover has one or more radially spaced apart concentric indicator lines for monitoring the separation of the components. When the base and cover are superimposed, the first and second channels define an annular interior chamber having an off-centered figure eight configuration. The base and cover each comprise a grooved column axially centered and extending from the base top surface and the cover bottom surface, respectively. The flexible bag comprises inner and outer tubes extending from the core of the bag and which are seated in the column grooves to ensure that the bag is fixably rotatable with the rotor.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to novel methods, devices and apparatusesfor the centrifugal separation of a liquid into its components ofvarying specific gravities, and is more particularly directed toward ablood separation device useful, for example, in the separation of bloodcomponents for use in various therapeutic regimens.

[0003] 2. Description of the State of Art

[0004] Centrifugation utilizes the principle that particles suspended insolution will assume a particular radial position within the centrifugerotor based upon their respective densities and will therefore separatewhen the centrifuge is rotated at an appropriate angular velocity for anappropriate period of time. Centrifugal liquid processing systems havefound applications in a wide variety of fields. For example,centrifugation is widely used in blood separation techniques to separateblood into its component parts, that is, red blood cells, platelets,white blood cells, and plasma.

[0005] The liquid portion of the blood, referred to as plasma, is aprotein-salt solution in which red and white blood cells and plateletsare suspended. Plasma, which is 90 percent water, constitutes about 55percent of the total blood volume. Plasma contains albumin (the chiefprotein constituent), fibrinogen (responsible, in part, for the clottingof blood), globulins (including antibodies) and other clotting proteins.Plasma serves a variety of functions, from maintaining a satisfactoryblood pressure and providing volume to supplying critical proteins forblood clotting and immunity. Plasma is obtained by separating the liquidportion of blood from the cells suspended therein.

[0006] Red blood cells (erythrocytes) are perhaps the most recognizablecomponent of whole blood. Red blood cells contain hemoglobin, a complexiron-containing protein that carries oxygen throughout the body whilegiving blood its red color. The percentage of blood volume composed ofred blood cells is called the “hematocrit.”

[0007] White blood cells (leukocytes) are responsible for protecting thebody from invasion by foreign substances such as bacteria, fungi andviruses. Several types of white blood cells exist for this purpose, suchas granulocytes and macrophages which protect against infection bysurrounding and destroying invading bacteria and viruses, andlymphocytes which aid in the immune defense.

[0008] Platelets (thrombocytes) are very small cellular components ofblood that help the clotting process by sticking to the lining of bloodvessels. Platelets are vital to life, because they help prevent bothmassive blood loss resulting from trauma and blood vessel leakage thatwould otherwise occur in the course of normal, day-to-day activity.

[0009] If whole blood is collected and prevented from clotting by theaddition of an appropriate anticoagulant, it can be centrifuged into itscomponent parts. Centrifugation will result in the red blood cells,which weigh the most, packing to the most outer portion of the rotatingcontainer, while plasma, being the least dense will settle in thecentral portion of the rotating container. Separating the plasma and redblood cells is a thin white or grayish layer called the buffy coat. Thebuffy coat layer consists of the white blood cells and platelets, whichtogether make up about 1 percent of the total blood volume.

[0010] These blood components, discussed above, may be isolated andutilized in a wide range of diagnostic and therapeutic regimens. Forexample, red blood cells are routinely transfused into patients withchronic anemia resulting from disorders such as kidney failure,malignancies, or gastrointestinal bleeding and those with acute bloodloss resulting from trauma-or surgery. The plasma component is typicallyfrozen by cryoprecipitation and then slowly thawed to producecryoprecipitated antihemophiliac factor (AHF) which is rich in certainclotting factors, including Factor VIII, fibrinogen, von Willebrandfactor and Factor XIII. Cryoprecipitated AHF is used to prevent orcontrol bleeding in individuals with hemophilia and von Willebrand'sdisease. Platelets and white blood cells, which are found in the buffylayer component, can be used to treat patients with abnormal plateletfunction (thrombocytopenia) and patients that are unresponsive toantibiotic therapy, respectively.

[0011] Various techniques and apparatus have been developed tofacilitate the collection of whole blood and the subsequent separationof therapeutic components therefrom. Centrifugal systems, also referredto as blood-processing systems, generally fall into two categories,discontinuous-flow and continuous-flow devices.

[0012] In discontinuous-flow systems, whole blood from the donor orpatient flows through a conduit into the rotor or bowl where componentseparation takes place. These systems employ a bowl-type rotor with arelatively large (typically 200 ml or more) volume that must be filledwith blood before any of the desired components can be harvested. Whenthe bowl is full, the drawing of fresh blood is stopped, the whole bloodis separated into its components by centrifugation, and the unwantedcomponents are returned to the donor or patient through the same conduitintermittently, in batches, rather than on a continuous basis. When thereturn has been completed, whole blood is again drawn from the donor orpatient, and a second cycle begins. This process continues until therequired amount of the desired component has been collected.

[0013] Discontinuous-flow systems have the advantage that the rotors arerelatively small in diameter but have the disadvantage that theextracorporeal volume (i.e., the amount of blood that is out of thedonor at any given time during the process) is large. This, in turn,makes it difficult or impossible to use discontinuous systems on peoplewhose size and weight will not permit the drawing of the amount of bloodrequired to fill the rotor. Discontinuous-flow devices are used for thecollection of platelets and/or plasma, and for the concentration andwashing of red blood cells. They are used to reconstitute previouslyfrozen red blood cells and to salvage red blood cells lostintraoperatively. Because the bowls in these systems are rigid and havea fixed volume, however, it is difficult to control the hematocrit ofthe final product, particularly if the amount of blood salvaged isinsufficient to fill the bowl with red blood cells.

[0014] One example of a discontinuous-flow system is disclosed byMcMannis, et al., in his U.S. Pat. No. 5,316,540, and is a variablevolume centrifuge for separating components of a fluid medium,comprising a centrifuge that is divided into upper and lower chambers bya flexible membrane, and a flexible processing container bag positionedin the upper chamber of the centrifuge. The McMannis, et al., systemvaries the volume of the upper chamber by pumping a hydraulic fluid intothe lower chamber, which in turn raises the membrane and squeezes thedesired component out of the centrifuge. The McMannis, et al., systemtakes up a fairly large amount of space, and its flexible pancake-shapedrotor is awkward to handle. The McMannis, et al., system does not permitthe fluid medium to flow into and out of the processing bag at the sametime, nor does it permit fluid medium to be pulled out of the processingbag by suction.

[0015] In continuous-flow systems, whole blood from the donor or patientalso flows through one conduit into the spinning rotor where thecomponents are separated. The component of interest is collected and theunwanted components are returned to the donor through a second conduiton a continuous basis as more whole blood is being drawn. Because therate of drawing and the rate of return are substantially the same, theextracorporeal volume, or the amount of blood that is out of the donoror patient at any given time in the procedure, is relatively small.These systems typically employ a belt-type rotor, which has a relativelylarge diameter but a relatively small (typically 100 ml or less)processing volume. Although continuous-flow systems have the advantagethat the amount of blood that must be outside the donor or patient canbe relatively small, they have the disadvantage that the diameter of therotor is large. These systems are, as a consequence, large. Furthermore,they are complicated to set up and use. These devices are used almostexclusively for the collection of platelets.

[0016] Continuous-flow systems are comprised of rotatable and stationaryparts that are in fluid communication. Consequently, continuous-flowsystems utilize either rotary seals or a J-loop. A variety of types ofrotary centrifuge seals have been developed. Some examples of rotarycentrifuge seals which have proven to be successful are described inU.S. Pat. Nos. 3,409,203 and 3,565,330, issued to Latham. In thesepatents, rotary seals are disclosed which are formed from a stationaryrigid low friction member in contact with a moving rigid member tocreate a dynamic seal, and an elastomeric member which provides aresilient static seal as well as a modest closing force between thesurfaces of the dynamic seal.

[0017] Another rotary seal suitable for use in blood-processingcentrifuges is described in U.S. Pat. No. 3,801,142 issued to Jones, etal. In this rotary seal, a pair of seal elements having confrontingannular fluid-tight sealing surfaces of non-corrodible material areprovided. These are maintained in a rotatable but fluid-tightrelationship by axial compression of a length of elastic tubing formingone of the fluid connections to these seal elements.

[0018] Related types of systems which incorporate rotatable, disposableannular separation chambers coupled via rotary seals to stationarytubing members are disclosed in U.S. Pat. Nos. 4,387,848; 4,094,461;4,007,871; and 4,010,894.

[0019] One drawback present in the above-described continuous-flowsystems has been their use of a rotating seal or coupling elementbetween that portion of the system carried by the centrifuge rotor andthat portion of the system which remains stationary. While such rotatingseals have provided generally satisfactory performance, they have beenexpensive to manufacture and have unnecessarily added to the cost of theflow systems. Furthermore, such rotating seals introduce an additionalcomponent into the system which if defective can cause contamination ofthe blood being processed.

[0020] One flow system heretofore contemplated to overcome the problemof the rotating seal utilizes a rotating carriage on which a singlehousing is rotatably mounted. An umbilical cable extending to thehousing from a stationary point imparts planetary motion to the housingand thus prevents the cable from twisting. To promote the desired endsof sterile processing and avoid the disadvantages of adiscontinuous-flow system within a single sealed system, a family ofdual member centrifuges can be used to effect cell separation. Oneexample of this type of centrifuge is disclosed in U.S. Pat. No. RE29,738 to Adams entitled “Apparatus for Providing Energy CommunicationBetween a Moving and a Stationary Terminal”. As is now well known, dueto the characteristics of such dual member centrifuges, it is possibleto rotate a container containing a fluid, such as a unit of donatedblood and to withdraw a separated fluid component, such as plasma, intoa stationary container, outside of the centrifuge without using rotatingseals. Such container systems utilize a J-loop and can be formed asclosed, sterile transfer sets.

[0021] The Adams patent discloses a centrifuge having an outer rotatablemember and an inner rotatable member. The inner member is positionedwithin and rotatably supported by the outer member. The outer memberrotates at one rotational velocity, usually called “one omega,” and theinner rotatable member rotates at twice the rotational velocity of theouter housing or “two omega.” There is thus a one omega difference inrotational speed of the two members. For purposes of this document, theterm “dual member centrifuge” shall refer to centrifuges of the Adamstype.

[0022] The dual member centrifuge of the Adams patent is particularlyadvantageous in that, as noted above, no seals are needed between thecontainer of fluid being rotated and the nonmoving component collectioncontainers. The system of the Adams patent provides a way to processblood into components in a single, sealed, sterile system wherein wholeblood from a donor can be infused into the centrifuge while the twomembers of the centrifuge are being rotated.

[0023] An alternate to the apparatus of the Adams patent is illustratedin U.S. Pat. No. 4,056,224 to Lolachi entitled “Flow System forCentrifugal Liquid Processing Apparatus.” The system of the Lolachipatent includes a dual member centrifuge of the Adams type. The outermember of the Lolachi centrifuge is rotated by a single electric motorwhich is coupled to the internal rotatable housing by belts and shafts.

[0024] U.S. Pat. No. 4,108,353 to Brown entitled “Centrifugal ApparatusWith Oppositely Positioned Rotational Support Means” discloses acentrifuge structure of the Adams type which includes two separateelectrical motors. One electric motor is coupled by a belt to the outermember and rotates the outer member at a desired nominal rotationalvelocity. The second motor is carried within the rotating exteriormember and rotates the inner member at the desired higher velocity,twice that of the exterior member.

[0025] U.S. Pat. No. 4,109,855 to Brown, et al., entitled “Drive SystemFor Centrifugal Processing Apparatus” discloses yet another drivesystem. The system of the Brown, et al, patent has an outer shaft,affixed to the outer member for rotating the outer member at a selectedvelocity. An inner shaft, coaxial with the outer shaft, is coupled tothe inner member. The inner shaft rotates the inner member at twice therotational velocity as the outer member. A similar system is disclosedin U.S. Pat. No. 4,109,854 to Brown entitled “Centrifugal Apparatus WithOuter Enclosure”.

[0026] The continuous-flow systems described above are large andexpensive units that are not intended to be portable. Further, they arealso an order of magnitude more expensive than a standard,multi-container blood collection set. There exists the need, therefore,for a centrifugal system for processing blood and other biologicalfluids that is compact and easy to use and that does not have thedisadvantages of prior-art continuous-flow systems.

[0027] Whole blood that is to be separated into its components iscommonly collected into a flexible plastic donor bag, and the blood iscentrifuged to separate it into its components through a batch process.This is done by spinning the blood bag for a period of about 10 minutesin a large refrigerated centrifuge. The main blood constituents, i.e.,red blood cells, platelets and white cells, and plasma, havingsedimented and formed distinct layers, are then expressed sequentiallyby a manual extractor in multiple satellite bags attached to the primarybag.

[0028] More recently, automated extractors have been introduced in orderto facilitate the manipulation. Nevertheless, the whole process remainslaborious and requires the separation to occur within a certain timeframe to guarantee the quality of the blood components. This complicatesthe logistics, especially considering that most blood donations areperformed in decentralized locations where no batch processingcapabilities exist.

[0029] This method has been practiced since the widespread use of thedisposable plastic bags for collecting blood in the 1970's and has notevolved significantly since then. Some attempts have been made to applyhaemapheresis technology in whole blood donation. This techniqueconsists of drawing and extracting on-line one or more blood componentswhile a donation is performed, and returning the remaining constituentsto the donor. However, the complexity and costs of haemapheresis systemspreclude their use by transfusion centers for routine whole bloodcollection.

[0030] There have been various proposals for portable, disposable,centrifugal apparatus, usually with collapsible bags, for example as inU.S. Pat. Nos. 3,737,096, or 4,303,193 to Latham, Jr., or with a rigidwalled bowl as in U.S. Pat. No. 4,889,524 to Fell, et al. These devicesall have a minimum fixed holding volume which requires a minimum volumeusually of about 250 ml to be processed before any components can becollected.

[0031] U.S. Pat. No. 5,316,540 to McMannis, et al., discloses acentrifugal processing apparatus, wherein the processing chamber is aflexible processing bag which can be deformed to fill it with biologicalfluid or empty it by means of a membrane which forms part of the driveunit. The bag comprises a single inlet/outlet tubing for theintroduction and removal of fluids to the bag, and consequently cannotbe used in a continual, on-line process. Moreover, the processing baghas a the disadvantage of having 650 milliliter capacity, which makesthe McMannis, et al., device difficult to use as a blood processingdevice.

[0032] As discussed above, centrifuges are often used to separated bloodinto its components for use in a variety of therapeutic regimens. Onesuch application is the preparation of a bioadhesive sealant. Abioadhesive sealant, also referred to as a fibrin glue, is a relativelynew technological advance which attempts to duplicate the biologicalprocess of the final stage of blood coagulation. Clinical reportsdocument the utility of fibrin glue in a variety of surgical fields,such as, cardiovascular, thoracic, transplantation, head and neck, oral,gastrointestinal, orthopedic, neurosurgical, and plastic surgery. At thetime of surgery, the two primary components comprising the fibrin glue,fibrinogen and thrombin, are mixed together to form a clot. The clot isapplied to the appropriate site, where it adheres to the necessarytissues, bone, or nerve within seconds, but is then slowly reabsorbed bythe body in approximately 10 days by fibrinolysis. Important features offibrin glue is its ability to: (1) achieve haemostasis at vascularanastomoses particularly in areas which are difficult to approach withsutures or where suture placement presents excessive risk; (2) controlbleeding from needle holes or arterial tears which cannot be controlledby suturing alone; and (3) obtain haemostasis in heparinized patients orthose with coagulopathy. See, Borst, H. G., et al., J Thorac.Cardiovasc. Surg., 84:548-553 (1982); Walterbusch, G. J, et al., Thorac.Cardiovasc. Surg., 30:234-235 (1982); and Wolner, F. J, et al., Thorac.Cardiovasc. Surg., 30:236-237 (1982).

[0033] Despite the effectiveness and successful use of fibrin glue bymedical practitioners in Europe, neither fibrin glue nor its essentialcomponents fibrinogen and thrombin are widely used in the United States.In large part, this stems from the 1978 U.S. Food and DrugAdministration ban on the sale of commercially prepared fibrinogenconcentrate made from pooled donors because of the risk of transmissionof viral infection, in particular the hepatitis-causing viruses such asHBV and HCV (also known as non-A and non-B hepatitis virus). Inaddition, the more recent appearance of other lipid-enveloped virusessuch as HIV, associated with AIDS, cytomegalovirus (CMV), as well asEpstein-Barr virus and the herpes simplex viruses in fibrinogenpreparations makes it unlikely that there will be a change in thispolicy in the foreseeable future. For similar reasons, human thrombin isalso not currently authorized for human use in the United States. Bovinethrombin, which is licensed for human use in the United States isobtained from bovine sources which do not appear to carry significantrisks for HIV and hepatitis, although other bovine pathogens, such asbovine spongiform and encephalitis, may be present.

[0034] There have been a variety of methods developed for preparingfibrin glue. For example, Rose, et al. in U.S. Pat. No. 4,627,879discloses a method of preparing a cryoprecipitated suspension containingfibrinogen and Factor XIII useful as a precursor in the preparation of afibrin glue which involves (a) freezing fresh frozen plasma from asingle donor such as a human or other animal, e.g. a cow, sheep or pig,which has been screened for blood transmitted diseases, e.g. one or moreof syphilis, hepatitis or acquired immune deficiency syndrome, at about80° C. for at least about 6 hours, preferably for at least about 12hours; (b) raising the temperature of the frozen plasma, e.g. to betweenabout 0° C. and room temperature, so as to form a supernatant and acryoprecipitated suspension containing fibrinogen and Factor XIII; and(c) recovering the cryoprecipitated suspension. The fibrin glue is thenprepared by applying a defined volume of the cyroprecipitate suspensiondescribed above and applying a composition containing a sufficientamount of thrombin, e.g. human, bovine, ovine or porcine thrombin, tothe site so as to cause the fibrinogen in the suspension to be convertedto the fibrin glue which then solidifies in the form of a gel.

[0035] A second technique for preparing fibrin glue is disclosed by Marxin his U.S. Pat. No. 5,607,694. Essentially, a cryoprecipitate asdiscussed previously serves as the source of the fibrinogen componentand then Marx adds thrombin and liposomes. A third method discussed byBerruyer, (M.,) et al., entitled “Immunization by bovine thrombin usedwith fibrin glue during cardiovascular operations,” (J.) Thorac.Cardiovase. Surg., 105(5):892-897 (1992)) discloses a fibrin glueprepared by mixing bovine thrombin not only with human coagulantproteins, such as fibrinogen, fibronectin, Factor XIII, and plasminogen,but also with bovine aprotinin and calcium chloride.

[0036] The above patents by Rose, et al., and Marx, and the technicalpaper by Berruyer, et al. each disclose methods for preparing fibrinsealants; however, each of these methods suffer disadvantages associatedwith the use of bovine thrombin as the activating agent. A serious andlife threatening consequence associated with the use of fibrin gluescomprising bovine thrombin is that patients have been reported to have ableeding diathesis after receiving topical bovine thrombin. Thiscomplication occurs when patients develop antibodies to the bovinefactor V in the relatively impure bovine thrombin preparations. Theseantibodies cross-react with human factor V, thereby causing a factor Vdeficiency that can be sufficiently severe to induce bleeding and evendeath. See, Rapaport, S. I., et al., Am. (J.) Clin. Pathol., 97:84-91(1992); Berruyer, M., et al., J. Thorac. Cardiovasc. Surg., 105:892-897(1993); Zehnder, J., et al., Blood, 76(10):2011-2016 (1990); Muntean,W., et al., Acta Paediatr., 83:84-7 (1994); Christine, R. J., et al.,Surgery, 127:708-710 (1997).

[0037] Further disadvantages associated with the methods disclosed byMarx and Rose, et al. are that the cryoprecipitate preparations requirea large time and monetary commitment to prepare. Furthermore, great caremust be taken to assure the absence of any viral contaminants.

[0038] A further disadvantage associated with the methods previouslydisclosed is that while human thrombin is contemplated for use as anactivator, human thrombin is not available for clinical use and there isno evidence that patients will not have an antigenic response to humanthrombin. By analogy, recombinant human factor VIII has been shown toproduce antigenic responses in hemophiliacs. See, Biasi, R. de.,Thrombosis and Haemostasis, 71(5):544-547 (1994). Consequently, untilmore clinical studies are performed on the effect of human recombinantthrombin one cannot merely assume that the use of recombinant humanthrombin would obviate the antigenic problems associated with bovinethrombin. A second difficulty with thrombin is that it is autocatalytic,that is, it tends to self-destruct, making handling and prolongedstorage a problem.

[0039] Finally, as discussed above, fibrin glue is comprised primarilyof fibrinogen and thrombin thus lacking an appreciable quantity ofplatelets. Platelets contain growth factors and healing factors whichare assumed to be more prevalent in a platelet concentrate. Moreover,platelets aid in acceleration of the clotting process.

[0040] There is still a need, therefore, for a centrifugal system forprocessing blood and other biological fluids, that is compact and easyto use and that does not have the disadvantages of prior-artcontinuous-flow systems and furthermore there exists a need for aconvenient and practical method for preparing a platelet gel compositionwherein the resulting platelet gel poses a zero risk of diseasetransmission and a zero risk of causing an adverse physiologicalreaction.

[0041] There is also a widespread need for a system that, during bloodcollection, will automatically separate the different components ofwhole blood that are differentiable in density and size, with a simple,low cost, disposable unit.

[0042] There is further a need for a centrifugal cell processing systemwherein multiple batches of cells can be simultaneously and efficientlyprocessed without the use of rotational coupling elements.

[0043] There is yet a further need for a platelet concentrate that aidsin increasing the rate of fibrin clot formation, thereby facilitatinghaemostasis.

[0044] Preferably the apparatus will be essentially self-contained.Preferably, the equipment needed to practice the method will berelatively inexpensive and the blood contacting set will be disposableeach time the whole blood has been separated.

SUMMARY OF THE INVENTION

[0045] Accordingly, one object of this invention is to provide a methodand apparatus for the separation of components suspended or dissolved ina fluid medium by centrifugation. More specifically, one object of thisinvention is to provide a method for the separation and isolation of oneor more whole blood components, such as platelet rich plasma, whiteblood cells and platelet poor plasma, from anticoagulated whole blood bycentrifugation, wherein the components are isolated while the centrifugeis rotating.

[0046] Another object of this invention is to utilize the isolated cellcomponents in a therapeutic regimen.

[0047] Another object of this invention is to provide an apparatus forthe separation of whole blood components, wherein the apparatus containsa centrifuge bag that provides for simultaneous addition of whole bloodfrom a source container and the withdrawal of a specific blood componentduring centrifugation.

[0048] Another object of this invention is to provide disposable,single-use centrifuge bags for holding whole blood during the separationof components of the whole blood by centrifugation, wherein the bag isadapted for use in a portable, point-of-use centrifuge.

[0049] Another object of this invention is to provide a portablecentrifuge containing a disposable centrifuge bag that maximizes theamount of a predetermined blood fraction that can be harvested from analiquot of blood that is of greater volume than the capacity of thedisposable centrifuge bag.

[0050] To achieve the foregoing and other objects and in accordance withthe purposes of the present invention, as embodied and broadly describedtherein, one embodiment of this invention comprises a flexible,disposable centrifuge bag adapted to be rotated about an axis,comprising:

[0051] a) one or more tubes, and

[0052] b) upper and lower flexible sheets, each sheet having a doughnutshaped configuration, an inner perimeter defining a central core and anouter perimeter, wherein the upper and lower sheets are superimposed andcompletely sealed together at their outer perimeters, and wherein thetubes are sandwiched between the upper and lower sheets and extend fromthe central core toward the outer perimeter, such that when the upperand lower sheets are sealed at the inner perimeter the tubes are sealedbetween the upper and lower sheets at the inner perimeter and are influid communication with the environment inside and outside thecentrifuge bag. The one or more tubes are fluidly connected to anumbilical cable comprising one or more lumen equal to the number oftubes of the centrifuge bag.

[0053] To further achieve the foregoing and other objects of thisinvention, another embodiment of the present invention comprises a rigidmolded container adapted to be rotated about an axis, comprising arigid, annular body having an axial core that is closed at the top endand opened at the bottom end. The rigid molded container furthercomprises an interior collection chamber for receiving and holding afluid medium to be centrifuged, the chamber having an outer perimeter,an inner perimeter, and a generally off-centered “figure eight” shapedcross-sectional area. The rigid molded container further comprises afirst channel which extends radially from the core and is in fluidcommunication with a point near the outer perimeter of the chamber, anda second channel which extends radially from the core and is in fluidcommunication with an area near the narrow portion or “neck” of thefigure eight-shaped chamber. The first and second channels thus providefluid communication with the environment inside and outside the interiorcollection chamber. The first and second channels are fluidly connectedto a dual lumen tubing having an inlet lumen and an outlet lumen.

[0054] To further achieve the foregoing and other objects of thisinvention, another embodiment of the present invention is an apparatusand method for separating components contained in a fluid medium. Moreparticularly, the present invention utilizes the principles ofcentrifugation to allow for the separation of whole blood into fractionssuch as platelet rich plasma and platelet poor plasma. In one aspect ofthe present invention, the above-described separation of the componentsis provided by utilizing a rotatable centrifuge motor comprising a basehaving a central column and a disposable centrifuge bag having a centralcore and which is positionable within the centrifuge motor and rotatabletherewith. The disposable centrifuge bag, which holds the whole bloodduring centrifugation, further comprises an inlet tube for introducingthe whole blood to the centrifuge bag, and an outlet tube for removingthe desired blood fraction from the centrifuge bag. The inlet and outlettubes are in fluid communication with a dual lumen tubing. Thecentrifuge bag is removably fixed within the centrifuge rotor byinserting the raised column through the bag center core and securingwith the cover. During the rotation of the centrifuge, components of thewhole blood will assume a radial, horizontal position within thecentrifuge bag based upon a density of such components, and thus thefluid medium components will be separated from other components havingdifferent densities.

[0055] Once a desired degree of separation of whole blood has beenachieved, the present invention provides for the specific removal of thedesired fraction within one or more of the regions from the centrifugebag through the outlet tube during continued rotation of the centrifuge,thereby allowing for on-line removal of the desired fraction. Additionalaliquots may be added to the centrifuge bag via the inlet tubesimultaneously or after the desired component has been harvested. In oneembodiment, the centrifuge bag is a flexible, transparent, generallyflat doughnut-shaped bag. In another embodiment, the centrifuge bag is arigid, transparent container having an interior chamber for receivingand holding the fluid medium during centrifugation, the interior chamberhaving a generally off-centered figure eight cross-sectionalconfiguration.

[0056] Another aspect of the present invention comprises a disposablecentrifuge bag having an inlet tube and an outlet tube, wherein theoutlet tube is fluidly connected with a bent fitting.

[0057] Another aspect of the present invention comprises a centrifugerotor for holding a centrifuge bag, the rotor comprising a base and acover, the base further having a first grooved, raised center column andthe cover having a second grooved, raised center column. The centrifugebag is a flexible, doughnut-shaped bag comprising inlet and outlet tubesin fluid communication with the environment inside and outside thecentrifuge bag, wherein the tubes are seated in the base and covercolumn grooves to hold the centrifuge bag in a fixed position relativeto the base and cover, such that the bag does not spin independently ofthe base and cover but rather spins concurrently and at the same rate ofrotation as the base and cover.

[0058] Another aspect of the present invention comprises a centrifugerotor for holding a centrifuge bag, the rotor comprising a base and acover for securing a centrifuge bag therebetween, the centrifuge coverfurther comprising one or more concentric indicator circles that arespaced from the center of the cover or the base to aid the operator invisualizing the distal ends of these tubes.

[0059] Another aspect of the present invention for the separation ofcomponents of a fluid medium (e.g., whole blood) utilizes a centrifugerotor comprising an interior chamber having a complex configuration,wherein the chamber holds a flexible, doughnut-shaped centrifuge bag forretaining the fluid medium during centrifugation. The centrifuge rotoris defined by a base having a lower chamber, and a cover having an upperchamber. When the cover is superimposed on the base, the upper and lowerchambers define the annular interior chamber of the rotor. The interiorrotor chamber has a generally off-centered figure eight-shapedcross-sectional configuration specifically designed to maximize thecollection of the desired component (e.g., platelet rich plasma) bycentrifugation of a fluid medium (e.g., anticoagulated whole blood). Thecentrifuge bag is formed from a substantially flexible material, suchthat the profile of the centrifuge bag during centrifugation is thusdetermined at least in part by the volume of the fluid medium containedtherein. When the centrifuge bag is filled to maximum capacity, itassumes the configuration of the interior of the rotor chamber.

[0060] Another aspect of this invention comprises a method for on-lineharvesting of a predetermined component of a fluid medium. Oneembodiment of the present invention utilizes a centrifuge and adisposable centrifuge bag for containing the fluid medium duringseparation and which is positionable within the centrifuge, thecentrifuge bag further comprising at least one inlet tube and at leastone outlet tube. The centrifuge includes a centrifuge rotor having abase portion, a cover, and an outer rim. The base portion and the coverdefine the interior of the centrifuge rotor, which is separated intoupper and lower chambers. The disposable centrifuge bag is positionablehorizontally within the lower chamber and may be appropriately securedto the centrifuge base by the cover. The centrifuge bag is fluidlyconnected via a dual lumen tubing to a source (e.g., to a containercomprising anticoagulated autologous whole blood) and collectioncontainer (e.g., for receiving platelet rich plasma or some othercomponent that will then be further processed). The dual lumen tubingcomprises an inlet lumen fluidly connected to the inlet tube of thecentrifuge bag and an outlet lumen fluidly connected to the outlet tubeof the centrifuge bag. The centrifuge bag is substantially annularrelative to the rotational axis of the centrifuge. When the centrifugebag is positioned within the centrifuge rotor and appropriately securedthereto to allow for simultaneous rotation, the fluid medium may beprovided to the centrifuge bag via the inlet lumen of the tubing duringrotation of the centrifuge. The components of the bag assume radial,horizontal positions base based on their densities. When a desireddegree of separation has been achieved, the desired fraction may beremoved from the centrifuge bag via the outlet lumen during continuedrotation of the centrifuge. The position of the fraction to be harvestedmay be shifted into the area of the outlet tube as needed, either bywithdrawing components that are positioned near the outer perimeterthrough the inlet tube, or by adding additional aliquots of the fluidmedium to the bag. In one embodiment of this method, the bag is aflexible, transparent doughnut-shaped bag. In another embodiment of thismethod, the bag is a rigid, transparent bag comprising an interiorchamber having an off-centered, figure eight cross-sectionalconfiguration.

[0061] It is yet another object of the invention to provide acentrifugal liquid processing system that may be automated.

[0062] It is yet another object of the present invention to provide acentrifuge having an internal lead drive mechanism allowing for acompact size.

[0063] A further object of the present invention is to provide for amethod and device for the production and isolation of thrombin for allmedical uses.

[0064] It is yet another object of this invention to provide a methodfor preparing a completely autologous platelet gel.

[0065] Another object of the present invention is to provide anautologous platelet gel wherein the risks associated with the use ofbovine and recombinant human thrombin are eliminated.

[0066] A further object of the present invention is to provide anautologous platelet gel for any application.

[0067] It is a further object of the present invention to providecellular components to be used in medical applications.

[0068] Additional objects, advantages, and novel features of thisinvention shall be set forth in part in the description and examplesthat follow, and in part will become apparent to those skilled in theart upon examination of the following or may be learned by the practiceof the invention. The objects and the advantages of the invention may berealized and attained by means of the instrumentalities and incombinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The accompanying drawings, which are incorporated in and form apart of the specifications, illustrate the preferred embodiments of thepresent invention, and together with the description serve to explainthe principles of the invention.

[0070] In the Drawings:

[0071]FIG. 1 is a perspective view illustrating one embodiment of thecontinuous-flow centrifugal processing system of the present inventionillustrating a centrifuge and side-mounted motor positioned within aprotective housing or enclosure of the invention.

[0072]FIG. 2 is an exploded side view of the centrifuge and theside-mounted motor of the centrifugal processing system of FIG. 1illustrating the individual components of the centrifuge.

[0073]FIG. 3 is a partial perspective view of the lower case assembly ofthe drive shaft assembly of FIG. 2.

[0074]FIG. 4 is an exploded side view of the lower case assembly of FIG.3.

[0075]FIG. 5 is an exploded perspective view of the components of thelower case assembly of FIG. 3.

[0076]FIG. 6 is a top view of the lower bearing assembly which ispositioned within the lower case assembly of FIG. 3.

[0077]FIG. 7 is a perspective view of the lower bearing assembly of FIG.6.

[0078]FIG. 8 is an exploded side view of the lower bearing assembly ofFIGS. 6 and 7.

[0079]FIG. 9 is a perspective view of the receiving tube guide of thecentrifuge of FIG. 2.

[0080]FIG. 10 is an exploded, perspective view of a gear of themid-shaft gear assembly of FIG. 2.

[0081]FIG. 11 is a perspective view of the gear of FIG. 10 as it appearsassembled.

[0082]FIG. 12 is an exploded, perspective view of the top bearingassembly of the centrifuge of FIG. 2.

[0083]FIG. 13 is a perspective view of the top case shell of the topbearing assembly of FIG. 12.

[0084]FIG. 14 is a perspective view of the centrifuge of the presentinvention shown in FIG. 1, having a quarter section cut away along lines14-14 of FIG. 1.

[0085]FIG. 15 is a perspective view of one embodiment of a centrifugerotor base.

[0086]FIG. 16 is a perspective view of one embodiment of a centrifugerotor cover.

[0087]FIG. 17 is a side cross-sectional view of one embodiment of arotor of this invention taken along view lines 17 of FIG. 14 for holdinga disposable centrifuge bag, showing a dual lumen tubing connected tothe bag.

[0088]FIG. 18 is a side cross-sectional view of one embodiment of arotor of this invention taken along view lines 18 of FIG. 1 for holdinga disposable centrifuge bag, showing the grooved columns of the base andcover.

[0089]FIG. 19 is an enlarged perspective view similar to FIG. 1illustrating an alternate embodiment of a centrifuge driven by aside-mounted motor (with only the external drive belt shown).

[0090]FIG. 20 is a cutaway side view of the centrifuge of FIG. 19illustrating the internal pulley drive system utilized to achieve adesired drive ratio and illustrating the rotor base configured forreceiving a centrifuge bag.

[0091]FIG. 21 is a cutaway side view similar to FIG. 20 with the rotorbase removed to better illustrate the top pulley and the location ofboth idler pulleys relative to the installed internal drive belt.

[0092]FIG. 22 is a sectional view of the centrifuge of FIG. 20 furtherillustrating the internal pulley drive system an showing the routing ofthe centrifuge tube (or umbilical cable).

[0093]FIG. 23 is a top view of a further alternate centrifuge similar tothe centrifuge of FIG. 19 but including internal, separate bearingmembers (illustrated as four cam followers) that allows the inclusion ofguide shaft to be cut through portions of the centrifuge for positioningof the centrifuge tube (or umbilical cable).

[0094]FIG. 24 is a perspective view similar to FIG. 19 illustrating thecentrifuge embodiment of FIG. 23 further illustrating the guide slot andshowing that the centrifuge can be driven by an external drive belt.

[0095]FIG. 25 is a top view of a flexible, disposable centrifuge bag ofthis invention.

[0096]FIG. 26 is a perspective view of a flexible, disposable centrifugebag of this invention.

[0097]FIGS. 27, 28, 29, and 30 are illustrations of bent fittings ofthis invention having “T” shaped, “curved T” shaped, “L” shaped, and “J”shaped configurations, respectively.

[0098]FIG. 31 is an illustration of an inlet and/or outlet tube of thisinvention.

[0099]FIG. 32 is a top view of a disposable centrifuge bag of thisinvention after the centrifugation of whole blood, showing the separatedblood components.

[0100] FIGS. 33-39 are schematic illustrations of one method of thisinvention for separating whole blood components using a disposablecentrifuge bag of this invention.

[0101]FIG. 40 is a top view of an alternate embodiment of a disposablecentrifuge bag of the present invention having inner and outer chambers.

[0102]FIG. 41 is a top view of the disposable centrifuge bag shown inFIG. 34 illustrating movement of the red blood cell layer from the outerperimeter toward the inner perimeter.

[0103]FIG. 42 is a bottom view of an alternate embodiment of adisposable centrifuge bag of the present invention having inner andouter chambers in fluid communication with outlet and inlet ports.

[0104]FIG. 43 is a side cross-sectional view of a rigid disposablecentrifuge bag of this invention.

[0105]FIG. 44 is a schematic illustration of separated blood componentscontained in a centrifuge bag having an elliptical cross-sectional viewof the centrifuge bag shown in FIG. 43.

[0106]FIG. 45 is a side cross-sectional view of a rigid disposablecentrifuge bag of this invention.

[0107]FIG. 46 is a schematic illustration of the surface areas andvarious dimensions of the figure eight configuration as shown in FIG.45.

[0108]FIG. 47 is a schematic illustration of separated blood componentscontained in a centrifuge bag having a figure eight side cross-sectionalconfiguration.

[0109]FIG. 48 is a side cross-sectional view of an alternativeembodiment of an assembled centrifuge rotor of this invention comprisingthe rotor cover of FIG. 49 and the rotor base of FIG. 50.

[0110]FIG. 49 is a side cross-sectional view of an alternativeembodiment of a rotor cover of this invention.

[0111]FIG. 50 is a side cross-sectional view of an alternativeembodiment of a rotor base of this invention.

[0112]FIG. 51 is a perspective view of the rotor base of FIG. 50.

[0113]FIG. 52 is a perspective view of the rotor cover of FIG. 49.

[0114]FIG. 53 is a block diagram illustrating the components of acentrifugal processing system of the present invention.

[0115]FIG. 54 is a graph illustrating the timing and relationship oftransmission of control signals and receipt of feedback signals duringoperation of one embodiment of the automated centrifugal processingsystem of FIG. 53.

[0116]FIG. 55 is a side view of an alternative embodiment of theautomated centrifugal processing system of FIG. 53 showing a centrifugehaving a rotor wherein the reservoir extends over the outer diameter ofthe centrifuge portion that facilitates use of an externally-positionedsensor assembly.

[0117]FIG. 56 is a side view of a further alternative embodiment of theexternal sensor assembly feature of the centrifugal processing system ofthe invention without an extended rotor and illustrating the positioningof a reflector within the centrifuge.

[0118]FIG. 57 is a side view of yet another embodiment of the externalsensor assembly feature of the centrifugal processing system of theinvention illustrating a single radiant energy source and detectordevice.

[0119]FIG. 58 is a block diagram of a an automated centrifugalprocessing system, similar to the embodiment of FIG. 47, includingcomponents forming a temperature control system for controllingtemperatures of separated and processed products.

[0120]FIG. 59 is a perspective view of components of the temperaturecontrol system of FIG. 58.

[0121]FIG. 60 is schematic and sectional view of the dispenser of thepresent invention.

[0122]FIG. 61 is a flow diagram representing the method for isolatingplatelet rich plasma and platelet poor plasma for use in preparing aplatelet gel of the present invention.

[0123]FIG. 62 is a flow diagram representing the final portion of themethod for preparing a platelet gel of the present invention usingplatelet rich plasma as a starting material.

[0124]FIG. 63 is a flow diagram representing the final portion of themethod for preparing a platelet gel of the present invention usingplatelet poor plasma as a starting material.

[0125]FIG. 64 is a graphic representation of the effect that theserum-to-plasma ratio has on clotting times.

[0126]FIG. 65 graphically represents the effect of calcium addition onthe clotting times of platelet rich plasma and platelet poor plasma.

[0127]FIG. 66 is a graphic representation of the relationship betweenclotting time and actual gel time using blood drawn from a donor.

[0128]FIG. 67 is a graphic representation of the relationship betweenclotting time and actual gel time using blood drawn from a donor.

[0129]FIG. 68 graphically represents the effect of calcium addition onclotting times and gel times using blood drawn from a donor.

[0130]FIG. 69 graphically represents the effect of calcium addition onclotting times and gel times using blood drawn from a donor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0131] The centrifugal processing system 10 of the present invention isbest shown in FIG. 1 having a stationary base 12, a centrifuge 20rotatably mounted to the stationary base 12 for rotation about apredetermined axis A, a rotor 202 for receiving a disposable bag (notshown) designed for continuous-flow. As illustrated, the centrifugalprocessing system 10 includes a protective enclosure 11 comprising themain table plate or stationary base 12, side walls 13, and a removablelid 15 made of clear or opaque plastic or other suitable materials toprovide structural support for components of the centrifugal processingsystem 10, to provide safety by enclosing moving parts, and to provide aportable centrifugal processing system 10. The centrifugal processingsystem 10 further includes a clamp 22 mounted over an opening (notshown) in the lid 15. Clamp 22 secures at a point at or proximately toaxis A without pinching off the flow of fluid that travels throughumbilical cable 228. A side mounted motor 24 is provided and connectedto the centrifuge 20 by way of a drive belt 26 for rotating the driveshaft assembly 28 (see FIG. 2) and the interconnected and driven rotorassembly 200 in the same rotational direction with a speed ratioselected to control binding of umbilical cable 228 during operation ofthe system, such as a speed ratio of 2:1 (i.e., the rotor assembly 200rotates twice for each rotation of the drive shaft assembly 28). Thepresent invention is further directed toward a dispensing device 902,best shown in FIG. 60 for the withdrawal and manipulation of specificblood components for various therapeutic regimens, such as but notlimited to the production of platelet rich plasma, platelet poor plasma,and white blood cells which may be used for the production of autologousthrombin and autologous platelet gels.

[0132] Referring now to FIG. 2, the continuous-flow centrifugalprocessing system 10 comprises a centrifuge 20 to which a rotor 202 isremovably or non-removably attached. The design of centrifuge 20 and itsself-contained mid-shaft gear assembly 108 (comprised of gears 110,110′, 131, and 74) is a key component of the invention thereby allowingfor the compact size of the entire centrifugal processing system 10 andproviding for a desired speed ratio between the drive shaft assembly 28and the rotor assembly 200.

[0133] The centrifuge 20 is assembled, as best seen in FIG. 2, byinserting the lower bearing assembly 66 into lower case shell 32 thusresulting in lower case assembly 30. Cable guide 102 and gears 110 and110′ are then positioned within lower case assembly 30, as will bediscussed in more detail below, so that gears 110 and 110′ are moveablyof engaged with lower bearing assembly 66. Upper bearing assembly 130 isthen inserted within top case shell 126 thus resulting in bearingassembly 124 which is then mated to lower case assembly 30, such thatgears 110 and 110′ are also moveably engaged with upper bearing assembly130, and held in place by fasteners 29. Lower bearing assembly 66 isjournaled to stationary base or main table plate 12 by screws 14, thusallowing centrifuge 20 to rotate along an axis A, perpendicular to maintable plate 12 (as shown in FIG. 1).

[0134] Referring now to FIGS. 3, 4, and 5, the lower case assembly 30 ispreferably, but not necessarily, machined or molded from a metalmaterial and includes a lower case shell 32, timing belt ring 46, timingbelt flange 50, and bearing 62 (e.g., ball bearings and the like). Lowercase shell 32 includes an elongated main body 40 with a smaller diameterneck portion 36 extending from one end of the main body 40 for receivingtiming belt ring 46 and timing belt flange 50. The larger diameter mainbody 40 terminates into the neck portion 36 thereby forming an externalshoulder 38 having a bearing surface 42 for timing belt ring 46. Timingbelt ring 46 and timing belt flange 50, as best seen in FIG. 5, haveinner diameters that are slightly larger than the outer diameter of neckportion 36 allowing both to fit over neck portion 36. Shoulder 38further contains at least one and preferably four internally threadholes 44 that align with hole guides 48 and 52 in timing belt ring 46and timing belt flange 50, respectively (shown in FIG. 5). Consequently,when assembled, screws 54 are received by hole guides 52 and 48 and arethreaded into thread holes 44 thus securing timing belt 46 and timingbelt flange 50 onto neck portion 36. Lower case shell 32 also has anaxial or sleeve bore 56 extending there through, and an internalshoulder 58, the upper surface 60 of which is in approximately the samehorizontal plane as external shoulder 38. Bearing 62 (shown in FIG. 4)is press fit concentrically into sleeve bore 56 so that it sits flushwith upper surface 60. Internal shoulder 58 also has a lower weightbearing surface 64 which seats on the upper surface 68 of lower bearingassembly 66, shown in FIGS. 6-8.

[0135] Lower bearing assembly 66 comprises a lower gear insert 70, ballbearings 84, gear 74 and spring pins 76 and 76′. As will become clear,the gear 74 may be of any suitable gear design for transferring an inputrotation rate to a mating or contacting gear, such as the gears 110,110′ of the mid-shaft gear assembly 108, with a size and tooth numberselected to provide a desired gear train or speed ratio when combinedwith contacting gears. For example, the gear 74 may be configured as astraight or spiral bevel gear, a helical gear, a worm gear, a hypoidgear, and the like out of any suitable material. In a preferredembodiment, the gear 74 is a spiral gear to provide a smooth toothaction at the operational speeds of the centrifugal processing system10. The upper surface 68 of lower gear insert 70 comprises an axiallypositioned sleeve 72, which receives and holds gear 74. gear 74 ispreferably retained within sleeve 72 by the use of at least one andpreferably two spring pins 76 and 76′ which are positioned within springpin holes 73 and 73′ extending horizontally through lower gear insert 70into sleeve 72. Thus, when gear 74 having spring pin receptacles 77 and77′ is inserted into sleeve 72 the spring pins 76 and 76′ enter thecorresponding receptacles 77 and 77′ thus holding the gear 74 in place.Of course, other assembly techniques may be used to position and retaingear 74 within the lower gear assembly 66 and such techniques areconsidered within the breadth of this disclosure. For example, gear 74may be held in sleeve 72 by a number of other methods, such as, but notlimited to being press fit or frictionally fit, or alternatively gear 74and lower gear insert 70 may be molded from a unitary body.

[0136] The base 78 of lower gear insert 70 has a slightly largerdiameter than upper body 80 of lower gear insert 70 as a result of aslight flare. This slight flare produces shoulder 82 upon which ballbearing 84 is seated. Once assembled lower bearing assembly 66 isreceived by sleeve bore 56 extending through neck portion 36 of lowercase shell 32. A retaining ring 86 is then inserted into the annularspace produced by the difference of the outer diameter of the lowerbearing assembly 66 and the inner diameter of sleeve bore 56 above ballbearings 84. A second retaining ring 87 (shown in FIG. 2) is alsoinserted into the annular space produced by the difference between theouter diameter of the lower bearing assembly 66 and the inner diameterof sleeve bore 56 below ball bearing 84, thereby securing lower gearinsert 70 within lower case shell 32. Consequently, ball bearings 62 and84 are secured by retaining rings 86 and 87, respectively, resulting inlower case shell 32 being journaled for rotation about lower bearingassembly 66 but fixed against longitudinal and transverse movementthereon. Therefore, when assembled lower bearing assembly 66 is mountedto stationary base 12, by securing screws 14 into threaded holes 79located in the base 78. Lower case shell 32 is thus able to freelyrotate about stationary lower bearing assembly 66 when the drive belt 26is engaged.

[0137] Referring now to FIG. 5, extending from the opposite end of neckportion 36 on lower case shell 32 are a number of protrusions or fingers88, 90, 92, and 94. Positioned between protrusions 88 and 90, andbetween protrusions 92 and 94 are recessed slots 96 and 98,respectively, for receiving tube guide 102 (FIG. 9). The function oftube guide 102 will be discussed in further detail below, but in shortit guides umbilical cable 228 connected to centrifuge bag 226 throughthe mid-shaft gear assembly 108 and out of the centrifuge 20.

[0138] Positioned between protrusions 90 and 92, and between protrusions88 and 94 are recessed slots 104 and 106, respectively, for receivinggears 110 and 110′ of mid-shaft gear assembly 108 (FIG. 2). The gears110 and 110′ are preferably configured to provide mating contact withthe gear 74 and to produce a desired, overall gear train ratio withinthe centrifuge 20. In this regard, the gears 110 and 110′ are preferablyselected to have a similar configuration (e.g., size, tooth number, andthe like) as the gear 74, such as a spiral gear design. As illustratedin FIGS. 2 and 14 mid-shaft gear assembly 108 comprises a pair of gears110 and 110′ engaged with gears 74 and 131. While the construction ofgears and gear combinations is well known to one skilled in themechanical arts, a brief description is disclosed briefly herein.

[0139]FIG. 10 illustrates an exploded view depicting the assembly ofgear 110, and FIG. 11 is a perspective view of the gear 110 of FIG. 10as it appears assembled. Gear 110′ is constructed in the same manner.Gear 111 is locked onto mid-gear shaft 112 using key stock 114 andexternal retaining ring 116. Ball bearing 118 is then attached to midgear shaft 112 using a flat washer 120 and cap screw 122. Recessed slots104 and 106 of lower case shell 32 then receive ball bearing 118 and118′ (not shown). In an alternate embodiment ball bearing 118 can bereplaced by bushings (not shown). When assembled, gears 110 and 110′make contact with the lower gear 74 (see FIGS. 2 and 14) to providecontact surfaces for transferring a force from the stationary gear 74 tothe gears 110 and 110′ to cause the gears 110 and 110′ to rotate at apredetermined rate that creates a desired output rotation rate for thedriven rotor assembly 200. The rotor assembly 200 is driven by the driveshaft assembly 28 which is rotated by the drive motor 24 at an inputrotation rate or speed, and in a preferred embodiment, the drive shaftassembly 28 through the use of the gears 110 and 110′ is configured torotate the rotor assembly 200 at an output rotation rate that is twicethe input rotation rate (i.e., the ratio of the output rotation rate tothe input rotation rate is 2:1). This ratio is achieved in theillustrated embodiment by locking the gears 110 and 110′ located withinthe drive shaft assembly 28 to rotate about the centrifuge center axis,A, with the lower case shell 32 which is rotated by the drive motor 24.The gears 110 and 110′ also contact the stationary gear 74 which forcesthe gears 110, 110′ to rotate about their rotation axes which aretraverse to the centrifuge center axis, A, and as illustrated, therotation axes of the gears 110, 110′ coincide. By rotating with thelower case shell 32 and rotating about the gear rotation axes, the gears110, 110′ are able to provide the desired input to output rotation rateof 2:1 to the rotor assembly 200.

[0140] In this regard, gears 110 and 110′ and tube guide 102 are lockedinto position by attaching top bearing assembly 124 to lower caseassembly 30. Top bearing assembly 124 (as shown in FIG. 12) comprisestop case shell 126, ball bearing 128, and an upper bearing 130. Top caseshell 126, as best seen in FIGS. 12 and 13, comprises an upper surface132, a lower lip 134 and a central or axial bore 136 there through.Upper surface 132 slightly overhangs axial bore 136 resulting in ashoulder 138 having a lower surface 140 (shown in FIG. 13). Lower lip134 is a reverse image of upper lip 100 on lower case shell 32 (shown inFIG. 5).

[0141] Upper bearing assembly 130 (FIG. 12) comprises an upper surface133 and a lower surface 135 wherein the upper surface 133 has a meansfor receiving a rotor 202. On the lower surface 135 a concentricallypositioned column 137 protrudes radially outward perpendicular to lowersurface 135. Upper bearing assembly 130 further comprises an axiallypositioned bore 139 that traverses column 137 and upper surface 133 andreceives upper gear insert 131. Upper gear insert 131 also contains anaxial bore 142 and thus when positioned concentrically within column 137axial bores 139 and 142 allow for umbilical cable 228 to travel throughupper bearing assembly 130 of top case shell 126 down to cable guide 102(shown in FIG. 14). As discussed previously with respect to lowerbearing assembly 66, upper gear insert 131 may be any suitable geardesign for receiving an input rotation rate from a mating or contactinggear, such as the gears 110, 110′ of the mid-shaft gear assembly 108,with a size and tooth number selected to provide a desired gear train orspeed ratio when combined with contacting gears. For example, gearinsert 131 may be configured as a straight or spiral bevel gear, ahelical gear, a worm gear, a hypoid gear, and the like. In a preferredembodiment, gear 131 is a spiral gear to provide a smooth tooth actionat the operational speeds of the centrifugal processing system 10. Gearinsert 131 is preferably retained within column 137 by use of at leastone and preferably two spring pins (not shown); however, other assemblytechniques may be used to position and retain the gear insert 131 withinthe column 137 and such techniques are considered within the breadth ofthis disclosure. For example, gear insert 131 may be held in column 137by a number of other methods, such as, but not limited to being pressfit or frictionally fit or alternatively gear insert 131 and the upperbearing assembly may be molded from a unitary body.

[0142] Upper bearing assembly 130 is then inserted into axial bore 136of top case shell 126 so that the lower surface 135 sits flush withupper surface 132 of top case shell 126. Ball bearing 128 is theninserted into the annular space created between the outer diameter ofcolumn 137 and the inner side wall 141 of top case shell 126 therebysecuring upper bearing assembly 130 into place.

[0143] Referring now to FIG. 13, lower lip 134 is contoured to mate withprotrusions 88, 90, 92 and 94 extending from lower case shell 32.Specifically, the outer diameter of lower lip 134 matches the outerdiameter of the upper end of main body 40 of lower case shell 32 andrecesses 144 and 148 receive and retain protrusions 88 and 92respectively, while recesses 146 and 150 receive and retain protrusions94 and 88, respectively. Holes are placed through each recess and eachprotrusion so that when assembled, fasteners 152 (shown in FIG. 12) canbe inserted through the holes thereby fastening the top bearing assembly124 to the lower case assembly 30.

[0144] Positioned between recesses 144 and 146 and between recesses 148and 150 are recessed slots 104′ and 106′, respectively, for receivinggears 110 and 110′ of mid-shaft gear assembly 108 (FIGS. 2 and 14). Thegears 110 and 110′ are preferably configured to provide mating contactwith the gear insert 131 and to produce a desired, overall gear trainratio within the centrifuge 20. In this regard, the gears 110 and 110′are preferably selected to have a similar configuration (e.g., size,tooth number, and the like) as the gear 131, such as a spiral geardesign. Furthermore recessed slots 96′ and 98′ exist between recesses144 and 150 and between recesses 146 and 148, respectively. When gears110 and 110′ are assembled as shown in FIG. 14, recessed slots 96 and96′ from the lower case shell 32 and top case shell 126, respectively,form port 154, and recessed slots 98 and 98′ form port 156 therebyallowing the umbilical cable 228 to exit centrifuge 20 through eitherport 154 or 156. Described above is one method of assembling thecentrifugal processing system 10 of the present invention; however,those skilled in the art will appreciate that the lower case assembly 30and upper bearing assembly can be joined in number of ways that allowthe four gears to be properly aligned with respect to one another.

[0145] In the above manner, the centrifugal processing system 10provides a compact, portable device useful for separating blood andother fluids in an effective manner without binding or kinking fluidfeed lines, cables, and the like entering and exiting the centrifuge 20.The compactness of the centrifugal processing system 10 is furthered bythe use of the entirely contained and interior gear train describedabove that comprises, at least in part, gear 74, gears 110 and 110′, andgear insert 131 of the upper bearing 130. The gear insert 131 of theupper bearing 130 is preferably selected to provide a contact surface(s)with the gears 110 and 110′ that transfers the rotation rate of thegears 110 and 110′ and consequently from gear 74 and to the gear insert131 of the upper bearing 130. In one preferred embodiment, the gearinsert 131 of the upper bearing 130 is a spiral gear rigidly mountedwithin the upper bearing 130 to rotate the rotor assembly 200 and havinga design similar to that of the spiral gear 74, i.e., same or similarface advance, circular pitch, spiral angle, and the like. Duringoperation, the gear 74 remains stationary as the lower case shell 32 isrotated about the centrifuge axis, A, at an input rotation rate, such asa rotation rate chosen from the range of 0 rpm to 5000 rpm. The gears110, 110′ are rotated both about the centrifuge axis, A, with the shell32 and by contact with the stationary gear 74. The spiral gears 110,110′ contact the gear insert 131 of the upper bearing 130 causing thegear insert 131 and connected upper bearing 130 to rotate at an outputrotation rate that differs, i.e., is higher, than the input rotationrate.

[0146] Although a number of gear ratios or train ratios (i.e., inputrotation rate/output rotation rate) may be utilized to practice theinvention, one embodiment of the invention provides for a gear trainratio of 1:2, where the combination and configuration of the gear 74,gears 110, 110′, and gear 131 of the upper bearing 130 are selected toachieve this gear train ratio. Uniquely, the rotation of the gears 110,110′ positively affects the achieved gear train ratio to allow, in oneembodiment, the use of four similarly designed gears which lowersmanufacturing costs while achieving the increase from input to outputrotation speeds. Similarly, as will be understood by those skilled inthe mechanical arts, numerous combinations of gears in differing number,size, and configuration that provides this ratio (or other selectedratios) may be utilized to practice the invention and such combinationsare considered part of this disclosure. For example, although two gears110, 110′ are shown in the mid-shaft gear assembly 108 to distributetransmission forces and provide balance within the operating centrifuge,more (or less) gears may be used to transmit the rotation of gear 74 tothe gear of the upper bearing 130. Also, just as the number, size, andconfiguration of the internal gears may be varied from the exemplaryillustration of FIGS. 1-14, the material used to fabricate the gear 74,the gears 110, 110′, and the gear insert 131 may be any suitable gearmaterial known in the art.

[0147] Another feature of the illustrated centrifugal processing system10 that advantageously contributes to compactness is the side-mounteddrive motor 24. As illustrated in FIGS. 1 and 2, the drive motor 24 ismounted on the stationary base 12 of the enclosure 11 adjacent thecentrifuge 20. The drive motor 24 may be selected from a number ofmotors, such as a standard electric motor, useful for developing adesired rotation rate in the centrifuge 20 of the centrifugal processingsystem 10. The drive motor 24 may be manually operated or, as in apreferred embodiment, a motor controller may be provided that can beautomatically operated by a controller of the centrifugal processingsystem 10 to govern operation of the drive motor 24 (as will bediscussed in detail with reference to the automated embodiment of theinvention). As illustrated in FIG. 1, a drive belt 26 may be used torotate the drive shaft assembly 28 (and, therefore, the rotor assembly200). In this embodiment, the drive belt 26 preferably has internalteeth (although teeth are not required to utilize a drive belt) selectedto mate with the external teeth of the timing belt ring 46 of the lowercase assembly 30 portion of the drive shaft assembly 28. The inventionis not limited to the use of a drive belt 26, which may be replaced witha drive chain, an external gear driven by the motor 24, and any othersuitable drive mechanisms. When operated at a particular rotation rate,the drive motor 24 rotates the drive shaft assembly 28 at nearly thesame rotation rate (i.e., the input rotation rate). A single speed drivemotor 24 may be utilized or in some embodiments, a multi and/or variablespeed motor 24 may be provided to provide a range of input rotationrates that may be selected by the operator or by a controller to obtaina desired output rotation rate (i.e., a rotation rate for the rotorassembly 200 and included centrifuge bag 226.

[0148] The present invention generally includes an apparatus and methodsfor the separation of a predetermined fraction(s) from a fluid mediumutilizing the principles of centrifugation. Although the principles ofthe present invention may be utilized in a plurality of applications,one embodiment of this invention comprises isolating predeterminedfraction(s) (e.g., platelet rich plasma or platelet poor plasma) fromanticoagulated whole blood. The platelet rich plasma may be used, forexample, in the preparation of platelet concentrate or gel, and moreparticularly may be used to prepare autologous platelet gel duringsurgery using blood drawn from the patient before or during surgery.

[0149] The centrifuge 20 has been discussed above and demonstrates thecompact and portable aspects of the present invention. To complete thedevice of the present invention a fluid collection device, also referredto as a bowl or rotor 202 is attached to the upper surface 133 of theupper bearing assembly 130 as shown in FIGS. 1 and 2. Rotor 202 ispreferably mounted permanently to upper bearing assembly 130, however,rotor 202 may also be capable of being removed. Rotor 202 comprises arotor base 204 (shown in FIG. 15) having a lower annular groove 212, anda rotor cover 206 having an upper annular groove 214. As shown in FIGS.17 and 18 the annular interior chamber 216 of rotor 202 is defined byupper and lower annular grooves 212 and 214. The lower annular 212receives a centrifuge bag 226 for containing the fluid medium to becentrifuged. Centrifuge bag 226 is connected to supply and receivingcontainers 398, 400, respectively, via umbilical cable 228 which ispreferably, but not limited to a dual lumen. There may be instanceswhere a certain technique requires multiple outlet or inlet ports andconsequently umbilical cable 228 of the present invention may comprisemultiple lumens. Umbilical cable 228 according to the preferredembodiment comprises inlet lumen 230 and outlet lumen 232 such that afluid medium may be provided to and removed from the centrifuge bag 226during rotation of the centrifuge rotor 202.

[0150] One embodiment of centrifuge rotor 202 is more particularlyillustrated in FIGS. 15, 16, 17 and 18. FIG. 15 is a perspective view ofrotor base 204, and FIG. 16 is a perspective view of rotor cover 206.FIG. 17 is a cross-sectional side view of rotor 202 taken along viewlines 17 in FIG. 1, and FIG. 18 is a cross-sectional side view of rotor202 taken along view lines 18 in FIG. 1. As illustrated in FIG. 15,rotor base 204 comprises raised annular rim 208 and raised column 218that is axially disposed in base 204. Raised column 218 further has agroove 222 extending across the diameter of column 218. Annular groove212 is defined by raised annular rim 208 and raised column 218. Theheight of rim 208 is equal to the height of column 218. Rotor cover 206shown in FIG. 16 comprises raised annular rim 210 and raised column 220which is axially disposed in rotor cover 206. Raised column 220 furtherhas a groove 224 extending across the diameter of column 220. Annulargroove 214 is defined by rim 210 and column 220. The height of rim 210is equal to the height of column 220.

[0151] Generally, when centrifuge rotor 202 is to be assembled for use,a flexible centrifuge bag such as a doughnut-shaped centrifuge bag 226(FIGS. 19 and 20) having a center core 242 is placed in rotor base 204such that center column 218 extends through the core 242 of centrifugebag 226 and the centrifuge bag 226 lies in annular groove 212. Rotorcover 206 is superimposed on rotor base 204 such that grooves 222 and224 are aligned, as illustrated in FIGS. 17 and 18. When rotor cover 204is secured to rotor base 206 by appropriate screws, fasteners, or thelike (not shown), rims 208 and 210 are in complete contact with eachother such that annular groove 212 and annular groove 214 define rotorinterior chamber 216. In one embodiment, columns 218 and 220 are incomplete contact with each other. Alternatively, the inner perimeter 240of centrifuge bag 226 is secured between columns 218 and 220 such thatcolumns 218 and 220 do not completely physically contact each other.

[0152] With the above description of one embodiment of the centrifuge inmind, another preferred embodiment of a centrifuge for use in thecentrifugal processing system 10 will be described. Referring to FIGS.19-22, a preferred embodiment of a centrifuge 640 is illustrated thatutilizes a uniquely arranged internal pulley system to obtain a desiredinput to output drive ratio (such as 2:1, as discussed above) ratherthan an internal gear assembly. The centrifuge 640 utilizes theside-mounted motor 24 (shown in FIG. 1) through drive belt 26 to obtainthe desired rotation rate at the rotor portion of the centrifuge.

[0153] Referring first to FIG. 19, the centrifuge 640 includes a rotorbase 644 (or top plate) with a recessed surface 648 for receiving andsupporting a centrifuge bag during the operation of the centrifuge 640.The rotor base 644 is rigidly mounted with fasteners (e.g., pins,screws, and the like) to a separately rotable portion (i.e., a toppulley 698 discussed with reference to FIGS. 20 and 21) of a lower caseshell 660. A cable port 656 is provided centrally in the rotor base 644to provide a path for a centrifuge tube or umbilical cable that is to befluidically connected to a centrifuge bag positioned on the recessedsurface 648 of the rotor base 644. It is important during operation ofthe centrifuge 640 to minimize and control contact and binding of theumbilical cable and moving parts (such as drive belts and pulleys). Inthis regard, the lower case shell 660 includes a side cable port 662 forthe umbilical cable to enter the centrifuge 640, which, significantly,the side cable port 662 is located between idler pulleys 666, 668 toprovide a spacing between any inserted tube or cable and the movingdrive components of the centrifuge 640.

[0154] Idler shaft or pins 664 are mounted and supported within thelower case shell 660 to allow the pins 664 to physically support thepulleys 666, 668. The idler pulleys 666, 668 are mounted on the pins 664by bearings to freely rotate about the central axis of the pins 664during operation of the centrifuge 640. The idler pulleys 666, 668 areincluded to facilitate translation of the drive or motive force providedor imparted by the drive belt 26 to the lower case shell 660 to therotor base 644, as will be discussed in more detail with reference toFIGS. 20 and 21, and to physically support the internal drive belt 670within the centrifuge 640. The drive belt 26 is driven by theside-mounted motor 24 (shown in FIG. 1) and contacts the lower caseshell 660 to force the lower case shell 660 to rotate about its centralaxis. The lower case shell 660 is in turn mounted on the base 674 in amanner that allows the lower case shell 660 to freely rotate on the base674 as the drive belt 26 is driven by the side-mounted motor 26. Thebase 674 is mounted to a stationary base 12 (shown in FIG. 1) such thatthe base 674 is substantially rigid and does not rotate with the lowercase shell 660.

[0155] Referring now to FIGS. 20-22, the centrifuge 640 is shown with acutaway view to more readily facilitate the discussion of the use of theinternal pulley assembly to obtain a desired output to input ratio, suchas two to one. As shown, the base 674 includes vibration isolators 676fabricated of a vibration absorbing material such as rubber, plastic,and the like through which the base 674 is mounted relatively rigidly tothe stationary base 12 (of FIG. 1). The drive belt 26 from theside-mounted motor 24 (of FIG. 1) contacts (frictionally or with the useof teeth and the like as previously discussed) a drive pulley 680, whichis rigidly mounted to the lower case shell 660. As the drive belt 26 isdriven by the motor 24, the lower case shell 660 through drive pulley680 rotates about its center axis (which corresponds to the center axisof the centrifuge 640). This rotation rate of the lower case shell 660can be thought of as the input rotation rate or speed.

[0156] To obtain a desired, higher rotation rate at the rotor base 644,the lower case shell 660 is mounted on the base to freely rotate aboutthe centrifuge center axis with bearings 690 that mate with the base674. The bearings 690 are held in place between the bottom pulley 692and the base 674, and the bottom pulley 692 is rigidly attached (withbolts or the like) to the base 674 to remain stationary while the lowercase shell 660 rotates. The illustrated bearings 690 are two piecebearings which allow the lower case shell 660 to rotate on the base 674.An internal drive belt 670 is provided and inserted through the lowercase shell 660 to contact the outer surfaces of the bottom pulley 692.The belt 670 preferably is installed with an adequate tension to tightlymate with the bottom pulley 692 such that frictional forces cause thebelt 670 to rotate around the stationary bottom pulley 692. Thisfrictional mating can be enhanced using standard rubber belts or beltswith teeth (and of course, other drive devices such as chains and thelike may be substituted for the belt 670).

[0157] The internal drive belt 670 passes temporarily outside thecentrifuge 640 to contact the outer surfaces of the idler pulleys 666and 668. These pulleys 666, 668 do not impart further motion to the belt670 but rotate freely on pins 664. The idler pulleys 666, 668 areincluded to allow the rotation about the centrifuge center axis by lowercase shell 660 to be translated to another pulley (i.e., top pulley 698)that rotates about the same axis. To this end, the idler pulleys 666,668 provide non-rigid (or rotable) support that assists in allowing thebelt 670 to be twisted without binding and then fed back into an upperportion of the lower case assembly 660 (as shown clearly in FIGS. 20 and21). As the internal drive belt 670 is fed into the lower case assembly660, the belt 670 contacts the outer surfaces of a top pulley 698.

[0158] During operation of the centrifuge 640, the movement of theinternal drive belt 670 causes the top pulley 698 to rotate about thecentrifuge center axis. The idler pulleys 666 and 668 by the nature oftheir placement and orientation within the centrifuge 640 relative tothe pulleys 692 and 698 cause the rotor base 644 to rotate in the samedirection as the lower case shell 660. Significantly, the top pulley 698rotated about the centrifuge center axis at twice the input rotationrate because it is mounted to the lower case shell 660 via bearings 694(preferably, a two piece bearing similar to bearings 690 but otherbearing configurations can be used) which are mounted to the centershaft 686 of the lower case shell 660 to frictionally contact an innersurface of the top pulley 698. Since the internal drive belt 670 isrotating about the bottom pulley 692 and the idler pulleys 666, 668 arerotating about the centrifuge central axis by drive belt 26, the toppulley 698 is turned about the centrifuge central axis in the samedirection as the lower case shell 660 but at twice the rate.

[0159] In other words, the drive force of the drive belt 26 and theinternal drive belt 670 are combined by the components of the centrifuge640 to create the output rotation rate. While a number of output toinput drive ratios may be utilized, as discussed previously, a 2:1 ratiois generally preferable, and the centrifuge 640 is preferably configuredsuch that the second, faster rotation rate of the top pulley 698 issubstantially twice that of the lower case shell 660. The use of aninternal drive belt 670 in combination with two pulleys rotating aboutthe same axis and the structural support for the pulleys within arotating housing results in a centrifuge that is very compact and thatoperates effectively at a 2:1 drive ratio with relatively low noiselevels (which is desirable in many medical settings).

[0160] The 2:1 drive ratio obtained in the top pulley 698 is in turnpassed on to the rotor base 644 by rigidly attaching the rotor base 644to the top pulley 698 with fasteners 652. Hence, a centrifuge bag placedon the recessed surface 648 of the rotor base 644 is rotated at a ratetwice that of the umbilical cable 228 that is fed into lower case shell660, which effectively controls binding as discussed above. The bearing694 (one or more pieces) wrap around the entire center shaft 686 of thelower case shell 660. To provide a path for the umbilical cord 228 topass through the centrifuge 640 to the rotor base 644 (which duringoperation will be enclosed with a rotor top or cover as shown in FIG.1), the rotor base 644 includes the cable port 656 and the center shaft686 is configured to be hollow to form a center cable guide. This allowsan umbilical cable 228 to be fed basically parallel to the centrifugecenter axis to the centrifuge bag (not shown). The lower case shell 660includes the side cable port 662 to provide for initial access to thecentrifuge 640 and also includes the side cable guide (or tunnel) 684 toguide the cable 228 through the lower case shell 660 to the hollowportion of the center shaft 686. The side port 662 and the side cableguide 684 are positioned substantially centrally between the two idlerpulleys 666, 668 to position the cable 228 a distance away from theinternal drive belt 670 to minimize potential binding and wear.

[0161] The centrifuge 640 illustrated in FIGS. 19-22 utilizes two piecebearings for both the bottom and top pulleys 692 and 698, respectively,and to provide a path for the umbilical cable 228 a central “blind”pathway (via side cable guide 684, the hollow center of the center shaft686, and cable ports 656, 662) was provided in the centrifuge 640. Whileeffective, this “blind” pathway can in practice present binding problemsas the relatively stiff cable 228 is fed or pushed through the pathway.To address this issue, an alternate centrifuge embodiment 700 isprovided and illustrated in FIGS. 23 and 24. In this embodiment, theupper portions of the centrifuge 700 include a guide slot between theidler pulleys 666, 668 that enables an umbilical cable 228 to be fedinto the centrifuge 700 from the top with the no components to block theview of the operator inserting the cable 228.

[0162] To allow a guide slot to be provided, the contiguous upperbearing 694 in the centrifuge 640 are replaced with bearing members thathave at least one gap or separation that is at least slightly largerthan the outer diameter of the cable 228. A number of bearing membersmay be utilized to provide this cable entry gap and are included in thebreadth of this disclosure. As illustrated, the centrifuge 700 includesa rotor base 702 that is rigidly fastened with fasteners 704 to the toppulley 698 (not shown) to rotate with this pulley at the output rate(e.g., twice the input rate) and to receive and support a centrifuge bagon recessed surface 716. The rotor base 702 further includes the cableport 718 which is useful for aligning the center of the bag and cable228 with the center of the centrifuge 700.

[0163] To allow ready insertion of the cable 228 in the centrifuge 700,the rotor base 702 further includes a cable guide slot 712 which asillustrated is a groove or opening in the rotor base 702 that allows thecable 228 to be inserted downward through the centrifuge 700 toward theside cable guide 724 of the lower case shell 720. The lower case shell720 also includes a cable guide slot 722 cut through to the top of theside cable guide 724. Again, the guide slots 712 and 724 are bothlocated in a portion of the centrifuge 700 that is between the idlerpulleys 666, 668 to position an inserted cable 228 from contacting andbinding with the internal drive belt 670, which basically wraps around180 degrees of the top pulley or lower case shell 720.

[0164] As shown in FIG. 23, the bearing members 706 are spaced apart andpreferably, at least one of these spaces or gaps is large enough to passthrough the cable 228 to the center shaft of the lower case shell 720.As illustrated, four cam followers are utilized for the bearing members706, although a different number may be employed. The cam followers 706are connected to the top pulley to enable the top pulley to rotate andare connected, also, to the center shaft of the lower case shell 720 torotate with the lower case shell 720. The cam followers 706 ride in abearing groove 710 cut in the lower case shell 720. To provide anunobstructed path for the cable 228, the cable guide slots 712 and 722are positioned between the two cam followers 706 adjacent the idlerpulleys 666, 668, and preferably the guide slots 712, 722 are positionedsubstantially centrally between the pulleys 666, 668. The guide slots712, 722 are positioned between these cam followers 706 to position thecable 228 on the opposite side of the centrifuge 700 as the contactsurfaces between the internal drive belt 670 and the top pulley 698(shown in FIGS. 20-22). In this manner, the use of separated bearingmembers 706 in combination with a pair of cable guide slots 712, 722allows an operator to readily install the umbilical cable 228 withouthaving to blindly go through the inside of the drive system andminimizes binding or other insertion difficulties.

[0165] A. Flexible, Disposable Centrifuge Bag

[0166] One embodiment of disposable, flexible centrifuge bag 226 is moreparticularly illustrated in FIGS. 25 and 26. The bag is an integral twostage self balancing disposable design. The disposable centrifuge bag226 has a substantially flat, toroidal- or doughnut-shaped configurationhaving outer and inner perimeters 238 and 240, respectively, andcomprises radially extending upper and lower sheets 234, 236 formed froma substantially flexible material. The upper and lower sheets 234, 236are superimposed and completely sealed together at outer perimeter 238by a heat weld, rf (radio frequency) weld or other comparable method ofadhering two surfaces. Inner perimeter 240 defines core 242 of bag 226.In one embodiment of the invention, centrifuge bag 226 further comprisesan inlet tube 248 sandwiched between upper and lower sheets 234, 236 andextending from the center of core 242 defined by inner perimeter 240 tothe outer perimeter 238 and an outlet tube 250 sandwiched between upperand lower sheets 234, 236 and extending from the center of the core 242to the outer perimeter 238. When upper and lower sheets 234, 236 aresealed together at inner perimeter 240, inlet and outlet tubes 248, 250are thereby sealed therebetween. Inlet and outlet tubes 248, 250 areeach in fluid communication with the interior of centrifuge bag 226 andthe environment outside centrifuge bag 226. The length of outlet tube250 is shorter than the length of inlet tube 248.

[0167] In one embodiment of this invention, outlet tube 250 is astraight tube as shown in FIG. 31. Alternatively, outlet tube 250includes a bent fitting 252 fluidly connected to the distal end ofoutlet tube 250 (FIGS. 25 and 26). The bent fitting 252 may be of anynumber of configurations, although preferably bent fitting 252 is shapedin the form of a “T”, “curved T”, a “J”, or an “L”, as illustrated inFIGS. 27, 28, 29 and 30, respectively. Alternatively, outlet tube 250and bent fitting 252 may be one contiguous molded unit rather than twoconnected pieces. Preferably, bent fitting 252 is in the shape of a “T”or a “curved T” as illustrated in FIGS. 27 and 28, respectively. The “T”or “curved T” design of bent fitting 252 ensures that the desired bloodcomponent (fraction) will be removed from the sides of the bent fitting252, rather than from a fraction located above or below the bentfitting, as discussed below in detail.

[0168] When the centrifuge bag 226 is positioned in the annular groove212 of the centrifuge rotor 202 as described above, it is critical thatinlet and outlet tubes 248, 250 are seated in groove 222. Further, whenrotor cover 206 is positioned over and removably secured to thecentrifuge base 204, it is important that inlet and outlet tubes 248,250 are also seated in groove 224. Seating inlet and outlet tubes 248,250 in grooves 222, 224 ensures that centrifuge rotor 202 is held in afixed position between rotor base 204 and rotor cover 206 such that thecentrifuge bag 226 and centrifuge rotor 202 rotate together. That is,the fixed position of centrifuge bag 226 ensures that centrifuge bag 226will not rotate independently of centrifuge bag 226 duringcentrifugation.

[0169] Inlet and outlet tubes 248, 250 are fluidly connected at theirproximal ends to umbilical cable 228, which in this particularembodiment is a dual lumen tubing connecting centrifuge bag 226 tosource and receiving containers 398, 400, respectively, for theintroduction and removal of components from the centrifuge bag 226during centrifugation (see FIG. 17). Dual lumen tubing 228 comprisesinlet lumen 230, which connects inlet tube 248 of centrifuge bag 226with source container 398, and outlet lumen 232, which connects outlettube 250 centrifuge bag 226 with receiving container 400. In oneembodiment, the inlet and outlet tubes 248, 250 are adapted at theirproximal ends for inserting into the inlet and outlet lumens 230 and232, respectively. Alternatively, connecting means 254 are inserted intothe proximal ends of inlet and outlet tubes 248, 250 for connecting thetubes to the inlet and outlet lumens 230, 232 as illustrated in FIG. 26.

[0170] In operation, one end of umbilical cable 228 must be secured torotor assembly 200 to prevent itself from becoming twisted duringrotation of rotor assembly 200 by the coaxial half-speed rotation ofdrive shaft assembly 28, which imparts a like rotation with respect tothe rotor 202 axis and consequently to the umbilical cable 228 that isdirected through cable guide 102. That is, if rotor assembly 200 isconsidered as having completed a first rotation of 360° and drive shaftassembly 28 as having completed a 180° half-rotation in the samedirection, the umbilical cable 228 will be subjected to a 180° twist inone direction about its axis. Continued rotation of rotor assembly 200in the same direction for an additional 360° and drive shaft assembly 28for an additional 180° in the same direction will result in umbilicalcable 228 being twisted 180° in the opposite direction, returningumbilical cable 228 to its original untwisted condition. Thus, umbilicalcable 228 is subjected to a continuous flexture or bending duringoperation of the centrifugal processing system 10 of the presentinvention but is never completely rotated or twisted about its own axis.

[0171] An alternative embodiment of a disposable centrifuge bag of thisinvention, shown in FIGS. 35 comprises two or more inlet tubes and/ortwo or more outlet tubes, wherein the tubes are fluidly connected to amultiple lumen tubing.

[0172] The disposable centrifuge bag 226 is formed from a transparent,substantially flexible material, including but not limited to, polyvinylchloride, polyethylene, polyurethane, ethylene vinyl acetate andcombinations of the above or other flexible materials. Based upon theflexibility of the centrifuge bag 226, the profile of the flexiblecentrifuge bag 226, shown in FIGS. 25 and 26, is determined at least inpart by the amount of fluid contained therein. The profile of centrifugebag 226 is further defined by the interior configuration of thecentrifuge rotor, as discussed below in detail. The ability tomanipulate the profile of centrifuge bag 226 based on the interiorconfiguration of the centrifuge rotor is utilized at least in part tomaximize the volume of fluid medium that can be contained in centrifugebag 226 during centrifugation, as will be discussed below.

[0173] The fluid or medium to be centrifuged may be contained withinsource container 300. For example, when the centrifuge 20 of thisinvention is used to prepare an autologous platelet gel, the fluid(i.e., whole blood), may be withdrawn from the patient during or priorto surgery into source container 398 containing an anticoagulant. Theanticoagulated whole blood is introduced to centrifuge bag 226 throughinlet tube 248 via inlet lumen 230 after the centrifuge bag 226 has beenpositioned in the centrifuge rotor 202 and rotation thereof isinitiated. As discussed above, securing centrifuge bag 226 in centrifugebase 204 in grooves 222, 224 holds the centrifuge bag 226 in a fixedposition therebetween, such that the centrifuge bag 226 cannot moveindependently of the centrifuge rotor 202, and therefore the centrifugebag 226 and rotor assembly 200 rotate concurrently at the same rate ofrotation. Rotation of the centrifuge rotor 202 directs the heavierdensity constituents of the anticoagulated whole blood within thecentrifuge bag 226 toward the outer perimeter 238 of the bag 226, whilethe lighter density constituents remain closer to an inner region, asillustrated in FIG. 32. More specifically, as illustrated in FIG. 32,when the fluid medium being separated is whole blood, the whole blood isseparated within centrifuge bag 226 into a red blood cell fraction(256), a white blood cell fraction (258), a platelet rich plasmafraction (260), and a platelet poor plasma fraction (262). As will beappreciated by those of skill in the art, whole blood fractions, redblood cell's and plasma are differently colored, and consequently theseparation of the fractions can be easily detected by the operator. Atan appropriate time during centrifuging, suction or other drawing meansmay be applied to the interior of centrifuge bag 226 via outlet lumen232 to remove the desired fraction from the centrifuge bag 226. In afurther embodiment, centrifuge cover 206 may further contain concentricindex lines to assist the operator in viewing the positions of outlettube 250 to the RBC plasma interface. Based on the speeds and times thelocation of the WBC and platelets can be varied with respect to the redblood cell's and plasma interface. For example, if the rpm is held low(approximately 1,000-1,700, preferably 1,500) the plasma and plateletswill separate from the RBC layer, as the rpm's are increased(1,400-1,700) the platelets will separate out of the plasma and resideat the plasma to RBC interface in greater concentrations. With increasedspeeds WBC reside deeper into the RBC pack.

[0174] With further regard to bent fittings 252, in one embodiment abent fitting is fluidly connected to the distal end of outlet tube 250.While bent fitting 252 is shown in FIG. 32 as having a “T” shape (FIG.27), this is for illustrative purposes only. Thus, it will beappreciated that bent fitting 252 as shown in FIG. 32 could have anumber of other configurations, such as those shown in FIGS. 25-31. Thedesign of bent fitting 252 ensures that the desired component iswithdrawn (e.g., the platelet rich plasma fraction 260) with less riskof contamination from withdrawing a portion of the adjacent fraction258. Thus, in one embodiment, the desired fraction is withdrawn when itsposition overlaps with the position of bent fitting 252. Alternatively,the inlet tube 248 may be first used to draw off the red blood cellfraction 256, and when it is desirable to remove the predeterminedfraction from the centrifuge bag 226, the predetermined fraction isdrawn through bent fitting 252 and outlet tube 250 and directed toreceiving container 400 via outlet lumen 232.

[0175] With continued reference to FIG. 32, as the separation of thefluid medium is initiated by centrifugation, substantially annularregions having constituents of a particular density or range ofdensities begin to form. For purposes of illustration, the separation ofwhole blood will be discussed, and as shown in FIG. 32 four regions arerepresented, each of which contains a particular type of constituent ofa given density or range of densities. Moreover, it should beappreciated that there may be a given distribution of densities acrosseach of the regions such that the regions may not be sharply defined.Consequently, in practice the regions may be wider (e.g., a largerradial extent) and encompass a range of densities of constituents.

[0176] In the example of FIG. 32, the first region 256 is the outermostof the four regions and contains red blood cells. The second region 258contains white blood cells, which have a lower density than that of thered blood cells. The third region 260 contains the platelet rich plasmafraction, and the innermost region 262 contains the least dense plateletpoor plasma fraction. In one embodiment, it may be desired to harvestthe platelet rich plasma fraction in region 260. In order to remove theplatelet rich plasma fraction from the centrifuge bag 226, vacuum orsuction is provided via outlet lumen 232 to the centrifuge bag 226 toremove a desired portion of region 260. A portion of the fraction 260that is in the area of the bent fitting 252 is drawn through bentfitting 252 and into an appropriate one of the collection containers 400(FIG. 17).

[0177] More specifically, FIGS. 33-39 illustrate one method of thisinvention for the separation of whole blood components, which is adynamic process. FIG. 33 shows one portion of the centrifuge bag 226,illustrating the separation of the whole blood components after infusionof an aliquot of whole blood into centrifuge bag 226 and centrifugationfor approximately 60 seconds to 10 minutes at a rate of rotation between0 and 5,000 rpms. It will be understood by those of skill in the artthat faster speeds of rotation will separate the blood in a shorterprior of time.

[0178]FIG. 33 shows the four separated whole blood fractions, with thedenser fractions closer to outer perimeter 238, and the less densefractions closer to inner perimeter 240. While it is well-known thathematocrits (i.e., the volume of blood, expressed as a percentage, thatconsists of red blood cells) will vary among individuals, ranging fromapproximately 29%-68%, such variations are easily adjusted for as aresult of the novel design of centrifuge bag 226 and consequently willnot affect the isolation of any of the desired fractions as discussedbelow in detail. Thus, for illustrative purposes, it will be assumedthat centrifugation of an initial infusion of an aliquot ofanticoagulated whole blood will give the profile shown in FIG. 33. Inone embodiment, it is desired to harvest the platelet rich plasmafraction 260. This may be achieved by performing a batch separationprocess or a continuous separation process as described below.

[0179] In one embodiment of a batch separation process of this inventionfor harvesting the platelet rich plasma fraction 260, centrifuge bag 226has a design as shown in FIG. 32 wherein bent fitting 252 positionedapproximately in the area where a platelet rich plasma fraction 260 istypically found after centrifugation of an aliquot of whole blood. Thisapproximation is simplified by the placement of concentric indicatorlines 205, 207, and 209, (not shown) in the upper surface of rotor cover206, wherein the concentric lines 205, 207 and 209 correspondapproximately with the edges of regions 260, 258, and 256, respectively.Alternative, concentric lines similar to 205, 207 and 209 may bedirectly imprinted onto the surface of centrifuge bag 226.

[0180] After centrifugation of an aliquot of blood contained incentrifuge bag 226, a substantial portion of the platelet rich plasmafraction 260 is withdrawn from centrifuge bag 226 through bent fitting252 while centrifuge rotor 202 is still spinning. As the volume of theplatelet rich plasma fraction 260 is reduced upon withdrawal, theinnermost fraction 262 naturally moves in the direction of the outerperimeter 238 due to centrifugal force, as shown in FIG. 34. Thewithdrawal of platelet rich plasma fraction 260 is terminated at a pointwhere the platelet poor plasma fraction 262 is close to bent fitting 252and before any significant portion of platelet poor plasma fraction 262could be withdrawn through bent fitting 252, as shown in FIG. 34. Thispoint can be determined either visually by the operator by volume, or bya sensor, as described below in detail. After withdrawal of the desiredplatelet rich plasma fraction 260, inlet lumen 230 is disconnected fromthe whole blood source container 398 and connected to a disposalcontainer, after which the remaining fluid in centrifuge bag 226 isevacuated through inlet tube 248 and directed to the disposal container.The inlet lumen is then reconnected to the whole blood source container,and the above-described batch process is repeated as many times asrequired until the necessary quantity of the desired fraction isisolated.

[0181] Alternatively, the above-described process can be performed as acontinuous process wherein the step of disconnecting the inlet lumen 230from the whole blood source 398 can be avoided. The continuous processseparation of whole blood may be achieve by using a disposablecentrifuge bag 226′ as illustrated in FIGS. 39-39 comprising an inlettube 248 and three outlet tubes 245, 247 and 250, wherein the tubes areconnected to an umbilical cable comprising four lumens. Morespecifically, a disposable centrifuge bag for use in a continuousseparation of whole blood comprises inlet tube 248 connected via aninlet lumen to a whole blood source container, a first outlet tube 250connected to a first outlet lumen that is in turn connected to aplatelet rich plasma receiving container, a second outlet tube 245connected via a second outlet lumen to either a red blood cell receivingcontainer or a waste container and a third outlet tube 247 connected viaa third outlet lumen to a platelet poor plasma receiving container. Inthe continuous separation process, after withdrawal of the portion ofplatelet rich plasma or other cellular components as described abovewith reference to FIGS. 33 and 34. Centrifuge bag has the capacity toreceive an additional volume (aliquot) of whole blood. Consequently, asshown in FIG. 35 infusion of an aliquot of whole blood is reinitiatedthrough first inlet tube 248 with continued centrifugation until thecapacity of the centrifuge bag 226′ is reached. As a result of theadditional volume of blood, the profile of the blood fractions incentrifuge bag 226′ will approximately assume the profile shown in FIG.35. As can be seen in FIG. 35, the additional volume of blood results ina shift of the location of the blood fractions, such that the plateletrich plasma fraction 260 has shifted back into the area of the bentfitting 252, and the platelet poor plasma fraction 262 has shifted backtowards the inner perimeter 240 and away from the vicinity of the bentfitting 252. Additional platelet rich plasma 260 can now be removed fromcentrifuge bag 226′ through outlet tube 250 as shown in FIG. 35.

[0182] As described above, removal of an additional volume of theplatelet rich plasma fraction 260 results in a shift in the location ofthe platelet poor plasma fraction 262 closer to the outer perimeter 238and consequently closer to the vicinity of bent fitting 252, as shown inFIG. 36, at which point removal of platelet rich plasma is againtemporarily terminated.

[0183] Additional infusions of whole blood aliquots to centrifuge bag262′ and removal of platelet rich plasma (by shifting the position ofthe platelet rich plasma fraction 260 relative to the position of thebent fitting 252) as described above may be repeated a number of times.Eventually, however, the continued infusion of whole blood followed byremoval of only the platelet rich plasma fraction will necessarilyresult in a gradual increase in the volumes (and consequently thewidths) of the remaining blood fractions 256, 258 and 260 in centrifugebag 226′. In particular, the volume, and therefore the width, of the redblood cell fraction 256 will increase to the extent that the otherfractions are pushed closer to the inner perimeter 240 (FIG. 37). Asshown in FIG. 37, the increased volume of red blood cells now present incentrifuge bag 226′ shifts the location of the fractions towards theinner perimeter 240 such that the white blood cell fraction 260 is nowin the vicinity of the bent fitting 252 as opposed to the desiredplatelet rich plasma fraction 262.

[0184] The novel design of centrifuge bag 226′ advantageously providesmeans for shifting the fractions back to the desired locations when thesituation shown in FIG. 37 arises. That is, second outlet tube 245serves as an inlet conduit for introduction of whole blood aliquots intocentrifuge bag 226′, also serves the function of withdrawing fractionsthat are located close to the outer perimeter 238. This is achieved inpart by attaching the second outlet lumen to either a red blood cellreceiving container or a waste container having a suction means (e.g.,syringe, pump, etc.) As shown in FIG. 38, second outlet tube 245, havingits distal end close to outer perimeter 238, can be operated to withdrawa substantial volume of the red blood cell fraction 256, which in turnshifts the location of the remaining fractions 258, 260, 262. Thewithdrawal of the red blood cell fraction 256 may be monitored visuallyby the operator, or by other means such as a sensor. Alternatively, thepositions of the fractions may be shifted by withdrawing the plateletpoor plasma fraction 262 through third outlet tube 247, which isconnected via a third outlet lumen to a platelet poor plasma receivingcontainer.

[0185]FIG. 37 shows that, after withdrawal of a portion of the red bloodcell fraction 256, the centrifuge bag 226′ again has the capacity toreceive an additional volume of whole blood for centrifugation. Anadditional infusion of an aliquot of whole blood through inlet tube 248into the centrifuge bag 226′ of FIG. 37 and centrifugation will producethe profile illustrated in FIG. 39. The above-described steps may berepeated as needed until the desired amount of platelet rich plasma hasbeen harvested. All of the above-described steps occur while thecentrifuge rotor 202 is spinning.

[0186] The above-described continuous separation method was illustratedin terms of performing the whole blood infusion step and the plateletrich plasma harvesting step sequentially. An alternative embodimentinvolves performing the infusion and harvesting steps substantiallysimultaneously, that is, the platelet rich plasma fraction is withdrawnat approximately the same time as an additional aliquot of whole bloodis being added to the bag. This alternate embodiment requires that thecentrifuge rotor spin at a rate that results in almost immediateseparation of the blood components upon infusion of an aliquot of wholeblood.

[0187] As stated previously, all of the above-described steps may bemonitored either visually by the operator by volume, or by a sensor. Ifthe steps are to be visually monitored, centrifuge cover 206 may furtherinclude one or more concentric indicator circles 205, 207, 209 (shown inFIGS. 17 and 18) which may be spaced from the center of cover 206 atdistances approximately equal to the outer edges of regions 260, 258256, respectively, to aid the operator in visualizing the positions ofthese regions with respect to 252.

[0188] FIGS. 33-39 illustrate one embodiment of how the design ofcentrifuge bags 226 and 226′ permit the general locations of the variousblood fractions to be shifted to allow for continuous harvesting of adesired blood fraction without the risk of contaminating the harvestedblood fraction, and further allow for continual on-line harvesting of alarge volume (10 to 5 L's) of blood using a small, portable centrifugedevice comprising a 10 cc to 200 cc capacity disposable centrifuge bags226 and 226′.

[0189] For example, the design of centrifuge bag 226 having inlet tube248 and outlet tube 250 means that the desired component or fractionwill be withdrawn from centrifuge bag 226 only through outlet tube 250,while the addition of whole blood aliquots or the removal of othercomponents (e.g., red blood cell fraction 256) will proceed only throughdual functional inlet tube 248. In this respect, the harvested fraction(e.g., platelet rich plasma fraction 260) is never withdrawn throughinlet tube 248 which was previously exposed to other fluid media (e.g.,whole blood or red blood cells). Thus, the design of centrifuge bag 226offers a significant advantage over conventional centrifuge containerscomprising only one tube which serves to both introduce the fluid mediumto the container and to withdraw the harvested fraction from thecontainer.

[0190] Furthermore, because of its unique design, the use of centrifugebags 226 and 226′ are independent of composition of the whole blood tobe centrifuged. For example, as stated above, hematocrits (i.e., thepercent volume of blood occupied by red blood cells) vary fromindividual to individual, and consequently the profile illustrated inFIG. 32 will vary from individual to individual. That is, the width ofred blood cell fraction 256 may be wider or narrower, which in turn willresult in the platelet rich plasma fraction 260 being positioned furtheraway in either direction from bent fitting 252. However, as discussedabove in detail with particular reference to FIGS. 33-34, the design ofcentrifuge bags 226 and 226′ allow the location of the desired fractionto be shifted until it is in the region of bent fitting 252. Suchshifting can be brought about, for example using centrifuge bag 226, bywithdrawing the red blood cell fraction through inlet tube 248, or byadding whole blood aliquots through inlet tube 248.

[0191] An alternative embodiment of a disposable, flexible centrifugebag 270 is illustrated in FIG. 40. The disposable centrifuge bag 270 hasa substantially flat, toroidal- or doughnut-shaped configuration havingouter and inner perimeters 271 and 272, respectively, and comprisesradially extending upper and lower sheets 273, 274 formed from asubstantially flexible material. The upper and lower sheets 273, 274 aresuperimposed and completely sealed together at outer perimeter 271 by anrf weld, heat weld or other comparable method of adhering two surfaces.Inner perimeter 272 defines core 275 of centrifuge bag 270. In oneembodiment of the invention, centrifuge bag 270 further comprises inlettube 276 sandwiched between upper and lower sheets 273, 274 and radiallyextending from the center of core 275 to the outer perimeter 271, andoutlet tube 278 sandwiched between upper and lower sheets 273, 274 andextending across the diameter of core 275 and having first and seconddistal ends 280, 281. When upper and lower sheets 273, 274 are sealedtogether at inner perimeter 272, inlet and outlet tubes 276, 278 arethereby sealed therebetween. Inlet and outlet tubes 276, 278 are each influid communication with the interior of centrifuge bag 270 and theenvironment outside centrifuge bag 270. Inlet tube 276 and outlet tube278 are fluidly connected to umbilical cable 228 (not shown), which inthis particular embodiment is a dual lumen tubing. Inlet tube 276 isfluidly connected at its proximal end to umbilical cable 228, preferablyby an L-shaped connector (not shown), and outlet tube 278 is fluidlyconnected at its center to umbilical cable 228 via a T-shaped connector(not shown).

[0192] The disposable centrifuge bag 270 is formed from a transparent,substantially flexible material, including but not limited to, polyvinylchloride, polyethylene, polyurethane, ethylene vinyl acetate andcombinations of the above or other flexible materials.

[0193] Upper and lower sheets 273, 274 of centrifuge bag 270 are furthersealed at two portions between the outer perimeter and the innerperimeter. That is, centrifuge bag 270 further comprises a firstC-shaped seal 282 located between the outer and inner perimeters 271,272 and having an first concave indentation or well 283 on the concaveside of C-shaped seal 282, and a second C-shaped seal 284 locatedbetween the outer and inner perimeters 271, 272 and having an secondconcave indentation or well 285 on the concave side of C-shaped seal284. First and second C-shaped seals 282 and 284 are formed by sealingportions of upper and lower sheets 273, 274 together by methods known inthe art for sealing two surfaces, including but not limited to rf orheat welding. Ends 288 and 289 of first C-shaped seal 282 are bentinward towards the inner core 275, and likewise ends 290 and 291 ofsecond C-shaped seal 284 are bent inward towards the inner core 275.First and second C-shaped seals 282, 284 have their concave sides facingeach other such that the first and second indentations 283, 285 arediametrically opposed to each other. That is, when centrifuge bag 270 isviewed from the top as in FIG. 40, first and second C-shaped seals 282,284 are mirror images of each other. First and second C-shaped seals282, 284 together define an outer chamber 292 between the outerperimeter 271 and first and second C-shaped seals 282, 284, wherein theouter chamber 292 has a toroidal configuration and serves as a firstprocessing compartment. First and second C-shaped seals 282, 284together further define an inner chamber 293 between first and secondC-shaped seals 282, 284 and inner perimeter 272, wherein the innerchamber 293 has a toroidal configuration and serves as a secondprocessing compartment. The first and second C-shaped seals 282, 284 arepositioned such that ends 288 and 290 are directly opposite and spacedapart from each other to define a first channel 286 therebetween, andsuch that ends 289 and 291 are directly opposite and spaced apart fromeach other to define a second channel 287 therebetween, wherein thefirst and second channels 286, 287 are diametrically opposed and providefluid communication between the first processing compartment 292 and thesecond processing compartment 293. Inlet tube 276 extends through eitherchannel 286 or channel 287, and the first and second distal ends 280,281 of outlet tube 278 extend into first and second indentations 283,285, respectively.

[0194] Centrifuge bag 270 is removably secured between rotor base 204and rotor cover 206 of rotor 202 in a manner as described above so thatcentrifuge bag 270 is held in a fixed position relative to rotor base204 and rotor cover 206 during rotation of the centrifuge rotor 202. Aswill be appreciated by those of skill in the art, alternativeembodiments of rotor base 204 (FIG. 15) and rotor cover 206 (FIG. 16)will be required to accommodate the design of centrifuge bag 270. Thus,an alternate embodiment of rotor base 204 comprises raised column 218comprising first and second grooves which are perpendicular to eachother and extend the diameter of the raised base column 218, such thatwhen rotor 202 is assembled, inlet tube 276 and outlet tube 278 ofcentrifuge bag 270 are seated in the first and second grooves,respectively, of raised base column 218. Similarly, an alternateembodiment of cover 206 comprises raised column 220 comprising first andsecond grooves which are perpendicular to each other and extend thediameter of the raised cover column 220, such that when rotor 202 isassembled, inlet tube 276 and outlet tube 278 are further seated in thefirst and second grooves, respectively, of raised cover column 220.

[0195] As stated above, inlet and outlet tubes 276, 278 are fluidlyconnected to umbilical cable 228, which in this particular embodiment isa dual lumen tubing connecting centrifuge bag 270 to source andreceiving containers 398, 400, respectively, for the introduction of thefluid to be centrifuged in bag 270 and for the removal of one or more ofthe separated components from the centrifuge bag 270 during rotation ofthe centrifuge 20. Dual lumen tubing 228 comprises inlet lumen 230,which connects inlet tube 276 with source container 398, and outletlumen 232, which connects outlet tube 278 with receiving container 400.

[0196] The fluid or medium to be centrifuged using centrifuge bag 270may be contained within source container 398. For example, when thecentrifuge 20 of this invention is used to prepare an autologousplatelet gel, the fluid (i.e., whole blood), may be withdrawn from thepatient during or prior to surgery into source container 398 containingan anticoagulant. The anticoagulated whole blood is introduced tocentrifuge bag 270 through inlet tube 276 via inlet lumen 230 after thecentrifuge bag 270 has been positioned in the centrifuge rotor 202 androtation thereof is initiated.

[0197] Centrifuge bag 270 may be used for the separation and isolationof one or more components dissolved or suspended in a variety of fluidmedia, including, but not limited to, the separation of cellularcomponents from biological fluids. For example, centrifuge bag 270 isuseful for the concentration and removal of platelets from whole blood.Therefore, the following description of the separation of platelets fromwhole blood using centrifuge bag 270 is merely for purposes ofillustration and is not meant to be limiting of the use of bag 270. Theseparation of a fluid medium such as whole blood in centrifuge bag 270may be considered to be a two-stage separation process. The first stageof the separation of platelets from whole blood involves separation of aplatelet suspension from the red blood cells. The platelet suspension istypically plasma rich in platelets, and it is commonly referred to asplatelet-rich plasma (PRP). However, as used herein, the term “plateletsuspension” is not limited to PRP in the technical sense, but isintended to encompass any suspension in which platelets are present inconcentrations greater than that in whole blood, and can includesuspensions that carry other blood components in addition to platelets.The second stage of the separation comprises separating platelets fromthe platelet suspension to produce a platelet concentrate. As usedherein, the term “platelet concentrate” is intended to encompass avolume of platelets that results after a “platelet suspension” undergoesa subsequent separation step that reduces the fluid volume of theplatelet suspension. The platelet concentrate may be a concentrate thatis depleted of white blood cells and red blood cells.

[0198] With reference to FIG. 41, stage one of a whole blood separationprocess using centrifuge bag 270 begins with the introduction of analiquot of whole blood into centrifuge bag 270 via inlet tube 276 duringrotation of the centrifuge 20. As the aliquot of whole blood entersouter chamber 292 of centrifuge bag 270, it quickly separates radiallyunder the influence of centrifugal force into various fractions withinouter chamber 292 based on the densities of the components of the wholeblood, including an outermost fraction containing the red blood cellswhich pack along the outer perimeter 271 of centrifuge bag 270, and aninner fraction comprising the platelet suspension. The plateletsuspension after centrifugation of the first aliquot of whole blood isrepresented in FIG. 41 by ring 294. Continued infusion of whole bloodinto the first processing compartment 292 adds an additional volume ofred blood cells and consequently pushes the platelet suspension inwardas represented by ring 295. Additional infusions of whole blood willcontinue to push the platelet suspension further inward, as representedby rings 296 and 297 until the first processing compartment 292 issubstantially filled with red blood cells (the remainder of the volumebeing plasma) such that the platelet suspension is pushed throughchannels 286 and 287 into second processing compartment 293. Asdiscussed above, the ends 288, 289 and 290, 291 of C-shaped seals 282,284, respectively, bend inward, which both helps to funnel the plateletsuspension through channels 286, 287 and to minimize the amount of redblood cells that pass through channels 286, 287. The point at which thered blood cells are near the entrance of channels 286, 287 may bemonitored either visually or by a sensor, as described below in detail.At this point the infusion of additional aliquots of whole blood isterminated, and the second stage of the two-stage separation processbegins.

[0199] During stage two of the separation process, the plateletsuspension which was pushed through channels 286, 287 into the secondprocessing compartment 293 flow under the influence of centrifugal forcetowards positions within the second processing compartment 293 that havethe greatest radial distances, that is, towards concave wells 283, 285,where the platelets, being the higher density component of the plateletsuspension, begin to collect and pack. The platelets can then bewithdrawn from concave wells 283, 285 through outlet tube 278. In theabove-described two-stage process for the separation of a plateletsuspension from whole blood, the first and second C-shaped seals 282,284 thus serve as physical barriers between the red blood cells and theplatelets to facilitate the separation and collection of platelets fromwhole blood. First and second concave wells 283, 285 act as reservoirsfor containing the platelets as they are separated from the plateletsuspension in the second stage of the separation process.

[0200] After withdrawal of the platelets from the wells 283 and 285,inlet lumen 230 is disconnected from the whole blood source container,after which the remaining components in centrifuge bag 270 are evacuatedthrough inlet tube 276 by applying suction to inlet lumen 230 and aredirected to a disposable container. The inlet lumen 230 is thenreconnected to the whole blood source container, and the above-describedbatch process is repeated as many times as required until the desiredquantity of platelets has been harvested.

[0201] An alternative embodiment of a disposable, flexible centrifugebag having inner C-shaped seals is illustrated in FIG. 42 as centrifugebag 320. Disposable centrifuge bag 320 has a substantially flat,toroidal- or doughnut-shaped configuration having outer and innerperimeters 322 and 324, respectively, and comprises radially extendingupper and lower sheets 323, 325 formed from a substantially flexiblematerial. The upper and lower sheets 323, 325 are superimposed andcompletely sealed together at outer perimeter 322 by an rf weld, heatweld or other comparable method of adhering two surfaces. Innerperimeter 324 defines core 327 of bag 320.

[0202] Upper and lower sheets 323, 325 of centrifuge bag 320 are furthersealed at two portions between the outer perimeter and the innerperimeter. That is, centrifuge bag 320 further comprises a firstC-shaped seal 326 located between the inner and outer perimeters 322,324, and a second C-shaped seal 328 located between the inner and outerperimeters 322, 324. The first and second C-shaped seals 326, 328 havetheir concave sides facing each other such that when centrifuge bag 320is viewed from the top as in FIG. 36, first and second C-shaped seals326 and 328 are mirror images of each other. First and second C-shapedseals 326, 328 together define an outer compartment 348 between theouter perimeter 322 and first and second C-shaped seals 326 and 328,wherein the outer compartment 348 has a toroidal configuration. Firstand second C-shaped seals further define an compartment 350 betweenfirst and second C-shaped seals 326, 328 and inner perimeter 324,wherein the inner compartment 350 has a doughnut shaped configuration.The ends 330 and 332 of first C-shaped seal 326 are slightly curvedinward towards the inner core 327, and likewise ends 334 and 336 ofsecond C-shaped seal 328 are slightly curved inward towards the innercore 327. The first and second C-shaped seals 326 and 328 are positionedsuch that ends 330 and 334 of first and second C-shaped seals 326, 328,respectively, are directly opposite and spaced apart from each other,thereby defining first channel 335 therebetween, and such that ends 332and 336 of first and second seals 326 and 328, respectively, aredirectly opposite and spaced apart from each other, thereby definingsecond channel 337 therebetween, wherein the first and second channels335 and 337 are diametrically opposed. First and second channels 335 and337 provide fluid communication between the outer and innercompartments.

[0203] Centrifuge bag 320 further comprises an inlet port 340, in thelower sheet 325 for introducing fluid into outer compartment 348.Preferably the inlet port 340 is spaced 90 degrees from channel 335however it could also be positioned at an angle greater or less than 90degrees from channel 335. Centrifuge bag 320 further comprises first andsecond outlet ports 344, 346 in lower sheet 325 and positioned withinchannels 335 and 337 for withdrawing a fluid compartment from centrifugebag 320.

[0204] In a preferred embodiment, centrifuge bag 320 comprises inlettube 338 secured to the outside surface of upper sheet 323 or lowersheet 325 and radially extending from the center of core 327 towards theouter perimeter 322, wherein inlet tube 338 is fluidly connected at itsdistal end to inlet port 340. Inlet port 340 fluidly connects inlet tube338 with the outer chamber 348 of centrifuge bag 320. Inlet tube 338 isfluidly connected at its proximal end to umbilical cable 228, preferablyby an L-shaped connector (not shown). Further, in a preferred embodimentcentrifuge bag 320 comprises outlet tube 342 secured to the outsidesurface of upper sheet 323 or lower sheet 325 and extending across thediameter of core 327, wherein one end of outlet tube 342 is fluidlyconnected to first outlet port 344 and the other end of outlet tube 342is fluidly connected to second outlet port 346. Outlet tube is fluidlyconnected at its center to umbilical cable 228 via a T-shaped connector(not shown).

[0205] In an alternative embodiment of this invention, centrifuge bag320 comprises inlet tube 338 sandwiched between upper and lower sheets323, 325 and extending radially from the center of core 327 towardsouter perimeter 322, wherein inlet tube 328 is fluidly connected at itsdistal end to inlet port 340, and outlet tube 342 sandwiched betweenupper and lower sheets 323, 325 and extending across the diameter ofcore 327, wherein one end of outlet tube 342 is fluidly connected tooutlet port 344 and the other end of outlet tube 342 is fluidlyconnected to outlet port 346. When upper and lower sheets 323, 325 aresealed together at inner perimeter 324, inlet and outlet tubes 338, 342are thereby sealed therebetween. Inlet tube 338 and outlet tube 342 arefluidly connected to umbilical cable 228 (not shown), which in thisparticular embodiment is a dual lumen tubing.

[0206] Centrifuge bag 320 is removably secured between rotor base 204and rotor cover 206 of rotor 202 in a manner as described above so thatcentrifuge bag 320 is held in a fixed position relative to rotor base204 and rotor cover 206 during rotation of the centrifuge rotor 202. Aswill be appreciated by those of skill in the art, alternativeembodiments of rotor base 204 (FIG. 15) and rotor cover 206 (FIG. 16) asdiscussed above with respect to centrifuge bag 270 will be required toaccommodate the design of centrifuge bag 370.

[0207] Centrifuge bag 370 may be used for the separation and isolationof one or more components dissolved or suspended in a variety of fluidmedia, including, but not limited to, the separation of cellularcomponents from biological fluids. For example, centrifuge bag 370is >useful for the concentration and removal of platelets from wholeblood. Therefore, the following description of the separation ofplatelets from whole blood using centrifuge bag 320 is merely forpurposes of illustration and is not meant to be limiting of the use ofbag 320. The separation of a fluid medium such as whole blood incentrifuge bag 320 may be considered to be a one-stage separationprocess. With reference to FIG. 36, centrifugation of whole blood beginswith the introduction of an aliquot of whole blood into centrifuge bag320 through inlet port 340 via inlet tube 338 during rotation of thecentrifuge 20. Inlet tube 338 is fluidly connected via inlet lumen 230of umbilical cable 228 to an anticoagulated whole blood source. As thealiquot of whole blood enters the outer chamber 348 of centrifuge bag320, it quickly separates radially within outer chamber 348 into variousfractions based on the densities of the components of the whole blood,including an outermost fraction containing the red blood cells whichpack along the outer perimeter 322 of centrifuge bag 320, and innerfractions containing the platelets and plasma. Continued infusion ofwhole blood adds an additional volume of red blood cells andconsequently pushes the fraction containing platelets inward. Additionalinfusions of whole blood will continue to push the platelet-containingfraction further inward until the chamber 348 is substantially filledwith red blood cells (the remainder of the volume being plasma), suchthat the platelet-containing fraction is pushed into channels 335, 337and into the vicinity of outlet ports 344, 346. As discussed above, theends of C-shaped seals 326, 328 curve slightly inward, which both helpsto funnel the platelet-containing fraction into channels 335, 337, andto minimize the amount of red blood cells that flow into channels 335,337. The point at which the red blood cells are near the entrance ofchannels 335, 337 may be monitored either visually or by a sensor, asdescribed below in detail. As the platelet-containing fraction entersthe vicinity of outlet ports 344, 346, the infusion of whole blood isterminated, and suction or other drawing means is applied to outlet tube342 to withdraw the platelet-containing fraction through outlet ports344, 346.

[0208] After withdrawal of substantial portion of the platelet richplasma, inlet lumen 230 is disconnected from the whole blood sourcecontainer and connected to a disposal container, after which theremaining components in centrifuge bag 320 are evacuated through inletport 34—by applying suction to inlet tube 276 and are directed to adisposal container. The inlet lumen 230 is then reconnected to the wholeblood source container, and the above-described process is repeated asmany times as required until the desired quantity of platelets has beenharvested.

[0209] B. Rigid Centrifuge Container

[0210] As can be appreciated, it may be desirable to maximize thesurface area of separated fraction to be harvested, since this maximizesthe amount of the fraction which may be collected without increasing thepotential for introducing impurities into the separation (e.g.,adjacent, lighter density components may begin moving into the region ofthe fraction being harvested), and without increasing the size of thecentrifuge to an undesirable degree.

[0211] In order to maximize the amount of the desired component (e.g.,platelet rich plasma, white blood cells, or platelet poor plasma) whichmay be harvested, one embodiment of a centrifuge container of thisinvention for the separation of components in a fluid medium (e.g.,whole blood), shown in FIGS. 45-52, is designed to position the desiredcomponent (e.g., platelet rich plasma) the platelet rich plasma at aregion within the fixed centrifuge container or centrifuge bag so thatthe desired fraction has a maximum horizontal surface area (i.e.,width). Thus, another embodiment of this invention comprises acentrifuge container 500 shown in FIG. 45. FIG. 45 is a sidecross-sectional view of a rigid container 500 comprising a rigid,annular body 510 having an axial core 600 that is closed at the top end610 and opened at the bottom end 620. Rigid container 500 furthercomprises an interior collection chamber 580 for receiving and holdingthe fluid medium to be centrifuged and having an outer perimeter 585 andan inner perimeter 590. The side, cross-sectional profile of chamber 580is generally an off-centered “figure eight” or “dumbbell” shape, asshown in FIG. 46. As used herein, “figure eight” or “dumbbell” shapedmeans that the height of section A is approximately equal to the heightof section C, and the heights of sections A and C are greater that theheight of section B. Furthermore, as used herein, “off-center” meansthat the width W₁ from the center of section B to outer perimeter 585 isless than the width W₂ from the center of section B to inner perimeter590 as shown in FIGS. 45 and 46.

[0212] Rigid container 500 further comprises inlet channel 550 extendingradially from core 600 to a point near the outer perimeter 585 and isfluidly connected at its distal end with the outer area of chamber 580.Rigid container 500 further comprises outlet channel 554 extendingradially from core 600 to the more central portion of chamber 580 (i.e.,the narrow portion or “neck” of the figure eight cross-section) and isfluidly connected at its distal end with chamber 580. While the inletand outlet channels 550, 554 are shown in FIG. 45 as being fluidlyconnected to the top end of chamber 580, the present invention alsoincludes embodiments wherein both channels 550, 554 are in fluidcommunication with the bottom end of chamber 580, or wherein channel 550is in fluid communication with the top end of chamber 580 and channel554 is in fluid communication with the bottom end of chamber 580, orvice versa. Inlet and outlet channels 550, 554 are fluidly connected todual lumen tubing 228 having an inlet lumen 230 and an outlet lumen 232.Rigid container 500 is removably secured to the upper surface 133 ofupper bearing assembly 130 with appropriate screws, fasteners or thelike (not shown). Inlet lumen 230 may be connected to a source for fluidmedium, and outlet lumen 232 may be connected to a suction means forwithdrawing the desired fraction from the chamber 580.

[0213] The configuration of chamber 580 is specifically designed tomaximize the collection of platelet rich plasma by centrifugation ofanticoagulated whole blood. More particularly, the shape of chamber 580increases the width of the platelet rich plasma fraction when viewedfrom the top and decreases the depth of the platelet rich plasmafraction when viewed from the side, thus allowing the withdrawal of agreater amount of platelet rich plasma. This unique design can be betterexplained by comparing FIGS. 44 and 47. FIG. 44 shows a side profile ofa rigid centrifuge container 500 as shown in FIG. 43, having a generallyoval profile and containing whole blood that has been separated intofour fractions by centrifugation. In FIG. 44, width W₃ indicates therelative horizontal width of the platelet rich plasma fraction to beharvested, and D₁ indicates the relative depth of the platelet richplasma fraction. FIG. 47 shows a side profile of rigid centrifugecontainer 580 of this invention having the above-described off-centeredfigure eight shape and containing whole blood that has been separatedinto four fractions by centrifugation. In FIG. 47, width W₄ indicatesthe relative horizontal width of the platelet rich plasma fraction 260to be harvested, and D₂ indicates the relative depth of the plateletrich plasma fraction 260. Width W₄ is necessarily wider than width W₃ inFIG. 44. Thus it can be easily appreciated that upon withdrawal of theplatelet rich plasma fraction 260 from the oval shaped container shownin FIG. 44, platelet poor plasma fraction 262 will shift closer to theoutlet tube 554 relatively quickly. In contrast the dumbbell shapedprofile of chamber 580 shown in FIG. 47 significantly increases thewidth W₄ while decreasing the average depth D₂, and therefore a greaterportion of the platelet rich plasma fraction 260 can be withdrawn withgreater accuracy before the platelet poor plasma fraction 262 reachesthe outlet tube 554.

[0214] In an embodiment where the platelet rich plasma is to becollected one could design chamber 580 as follows. The configuration ofchamber 580, that is, the relative heights A, B, and C as shown in FIG.46, will be determined based on the typical location of the plateletrich plasma fraction 260 after centrifugation of whole blood. Forexample, in a rigid centrifuge container 500 as illustrated in FIG. 45,having chamber 580 with a 30 ml capacity and a radius of approximately65 mm measured from its rotational axis to the edge 630, the plateletrich plasma will collect in chamber 580 at a region at a radial positionranging from about 35 to about 60 mm from the axis. In this region ofthe chamber 580, as illustrated in FIG. 46, the chamber 580 has a heightof about 10 mm such that the horizontal surface area “B” of this region,illustrated in FIG. 46, is about 4 mm². Consequently, it can beappreciated that because of the unique configuration of chamber 580, thesurface area of the platelet rich plasma fraction 260 as illustrated inFIG. 47 may be maximized without undesirably increasing the overall sizeof the rigid centrifuge container 500. It will be appreciated by thoseskilled in the art that various geometric designs may be utilizeddepending on the fluid medium being centrifuged and the cellularfraction to be collected. The process for harvesting platelets fromwhole blood using rigid container 500 may be achieved in a mannersimilar to that described for bag 226 Rigid centrifuge container 500 maybe made from any number of rigid, transparent materials that are capableof withstanding typical sterilization conditions, including but notlimited to acrylic resins, polycarbonate, or any clear thermal plastic.Preferably rigid container 500 is made of a cost-effective material thatis relatively inexpensive to dispose of.

[0215] C. Centrifuge Rotor Having a Complex Interior Geometry

[0216] An alternate embodiment of a centrifuge rotor of this inventionfor holding flexible centrifuge bag 226 is illustrated in FIGS. 48-52.Generally and referring to FIGS. 48 and 49, the centrifuge rotor 755 isdefined by a rotor base 760 (FIGS. 48, 50 and 52) having a lower channel780, and a rotor cover 770 (FIGS. 49 and 51) having an upper channel782. The annular interior chamber 784 (FIG. 48) of rotor 755 is definedby lower and upper channels 780, 782, and has a generally off-centeredfigure eight side cross-sectional configuration specifically designed tomaximize the collection of platelet rich plasma by centrifugation ofanticoagulated whole blood, as discussed below in detail.

[0217] As illustrated in FIGS. 51 and 52, rotor base 760 comprisesraised annular rim 775 and raised column 786 which is axially disposedin the interior of rotor base 760. Raised column 786 further has agroove 790 (FIG. 52) extending the diameter of column 786. The height ofrim 775 is equal to the height of column 786. As illustrated in FIG. 51,rotor cover 770 comprises raised annular rim 777 and raised column 788which is axially disposed in the interior of cover 770. Raised column788 further has a groove 792 (FIG. 52) extending the diameter of column788. The height of rim 777 is equal to the height of column 788. Rotorbase 760 and rotor cover 770 are preferably made from any number ofrigid transparent materials including, but not limited to acrylicresins, polycarbonate, or any clear thermal plastic.

[0218] When centrifuge rotor 755 is to be assembled for use, flexible,doughnut-shaped centrifuge bag 226 having a center core 242 is placed inrotor base 760 such that center column 786 preferably, but notnecessarily, extends through the core of centrifuge bag 226, and inletand outlet tubes 248, 250 of bag 226 are seated in groove 790. Rotorcover 770 is superimposed on rotor base 760 such that grooves 790 and792 are aligned and further so that inlet and outlet tubes 248, 250 areseated in groove 792. In one embodiment, when cover 770 is appropriatelysecured to base 760 (e.g., with screws, clamps, or the like), rims 775and 777 are in complete contact with each other, and columns 786 and 788are preferably in complete contact with each other, thereby creatingchamber 784 (FIG. 48). Alternatively when cover 770 is secured to base760 as described, the inner perimeter of bag 226 is secured betweencolumns 786 and 788 such that the columns do not physically contact eachother.

[0219] When the generally flat, flexible centrifuge bag 226 is containedwithin chamber 784 prior to the infusion of a fluid medium (e.g., wholeblood), it will not fill the entire volume of chamber 784 but ratherwill have a radially extending, flat shape as centrifuge rotor 755 isspinning. However, after a sufficient volume of the fluid medium (e.g.,whole blood) has been introduced into flexible bag 226 through inlettube 248 such that bag 226 is substantially completely filled, it willbe appreciated that filled centrifuge bag 226 will conform to the shapeof chamber 784 and consequently will have a off-centered figure eightshaped cross-section.

[0220] The off-centered figure eight configuration of the chamber 784 isof approximately the same configuration as the rigid bag 500. Therefore,for the same reasons, the shape of chamber 784 (and consequently theshape of filled bag 226), will assume an off-centered figure eight shapewherein the width of the platelet rich plasma fraction is greatlyincreased relative to the width of a filled bag having an ellipticalcross-sectional shape (see, for example, FIGS. 46 and 47).

[0221] As discussed above, a number of methods may be utilized to gaugethe harvesting of the desired fraction (such as, but not limited to,platelet rich plasma) from the centrifuge bag. For instance, theseparation of platelet rich plasma fraction may be indicated by visualobservation of a concentric ring containing the platelet rich plasma(which will be a less colored fraction) and an outer red-coloredconcentric ring containing the red blood cells. In this case, when suchfraction(s) have been separated, the platelet rich plasma may bewithdrawn from centrifuge bag 226 by bent fitting 160 to direct theplatelet rich plasma to the appropriate collector.

[0222] As an alternative to the foregoing, sensors may be incorporatedas discussed in detail below to detect the presence of the platelet richplasma fraction.

[0223] Based upon the foregoing, it can be appreciated that thecentrifugal processing system 10 and the centrifuge rotors and bags ofthis invention have a plurality of features which are suited toharvesting platelet rich plasma, white blood cells, platelet poor plasmaor red blood cells from a patient's whole blood in accordance with eachof the aspects of the present invention. For example, as discussedabove, hematocrits (the volume of blood occupied by red blood cells,expressed as a percentage) vary from individual to individual. Thus,depending on the amount of red blood cells present in a particularsample, the exact radial location of various blood components within thecentrifuge bag after centrifugation will also vary. The centrifuge bagsof this invention overcome this issue by having an inlet tube capable ofnot only introducing whole blood into the centrifuge bag, but alsocapable of withdrawing some of all of the red blood cell fraction asneeded to shift the location of the fraction to be harvested into thearea of the outlet tube. Such features are presented in centrifuge bags226, 270, 320 and 500. Yet another embodiment of the centrifuge bags ofthis invention which overcomes problems with varying hematocrits iscentrifuge bag 226′ having multiple outlet tubes.

[0224] Additionally, the centrifugal processing system 10 effectivelyprovides a closed system which enhances the potential for maintaining adesired degree of sterility associated with the entire procedure sincematerials can thus be both provided to and removed from the centrifugebag during rotation of the centrifuge via, for instance, a dual lumentubing connected to a fluid source (e.g., anticoagulated whole bloodwithdrawn from a patient before or during surgery) and collectioncontainers (i.e., for the preparation of a platelet gel), withoutinterrupting the process, and thus without significant exposure of thematerials to environmental conditions.

[0225] Moreover, the portable size of the centrifugal processing system10 in combination with the above-described features of shifting theseparated fractions and maximizing the surface area of the harvestedfraction allows for increased processing capabilities autologousplatelet gel over larger, conventional centrifuges

[0226] The on-line harvesting capabilities of the centrifugal processingsystem 10 allows for continuous, dynamic separation and collection ofplatelet rich plasma, white blood cells, red blood cells and plateletpoor plasma, by adjusting the input and removal of fluid medium andseparated fractions as described above. Further, the orientation of theflexible and rigid centrifuge bags of this invention and of the contentstherein (e.g., being generally radially extending) is not significantlymodified in the transformation from separation to harvesting of thevarious constituents. Moreover, vortexing throughout the contents of thecentrifuge bags of this invention is reduced or eliminated since thecentrifugal processing system 10 does not have to be decelerated orstopped for addition of fluid medium or removal of the various fractionstherefrom.

[0227] Further, the general orientation of the flexible and rigidcentrifuge bags of the invention (e.g., substantially horizontal) ismaintained during removal of the desired whole blood fraction similar tothe orientation of the centrifuge bags assumed during centrifugation tofurther assist in maintaining the degree of separation provided bycentrifugation. Consequently, the potential is reduced for disturbingthe fractions to the degree where the separation achieved is adverselyaffected.

[0228] Although the present invention has been described with regard tothe separation of whole blood components, it will be appreciated thatthe methods and apparatus described herein may be used in the separationcomponents of other fluid media, including, but not limited to wholeblood with density gradient media; cellular components, or sub-sets ofthe four whole blood components previously defined.

[0229] While blood separation and materials handling may be manuallycontrolled, as discussed above, a further embodiment of the presentinvention provides for the automation of at least portions of theseparation and material handling processes. Referring to FIG. 53, anautomated centrifugal processing system 800 is illustrated that isgenerally configured to provide automated control over the steps ofinputting blood, separating desired components, and outputting theseparated components. The following discussion of the processing system800 provides examples of separating platelets in a blood sample, but theprocessing system 800 provides features that would be useful forseparating other components or fractions from blood or other fluids.These other uses for the processing system 800 are considered within thebreadth of this disclosure. Similarly, the specific components discussedfor use in the processing system 800 are provided for illustrationpurposes and not as limitations, with alternative devices being readilyapparent to those skilled in the medical device arts.

[0230] In the embodiment illustrated in FIG. 53, the processing system800 includes a blood source 802 connected with a fluid line 804 to aninlet pump 810. A valve 806, such as a solenoid-operated valve or aone-way check valve, is provided in the fluid line 804 to allow controlof flow to and from the blood source 802 during operation of the inletpump 810. The inlet pump 810 is operable to pump blood from the bloodsource 802 through the fluid line 818 to a centrifuge 820. Once all or aselect portion of the blood in the blood source 802 have been pumped toa blood reservoir 824 of the centrifuge 820 the inlet pump 810 is turnedoff and the blood source 802 isolated with valve 806. The inlet pump 810may be operated at later times to provide additional blood during theoperation of the processing system 800 (such as during or after theremoval of a separated component).

[0231] The centrifuge 20 preferably includes a flexible centrifuge bag,for example 226, 226′, 270, or 320, positioned within the rotor 202 forcollecting the input blood, or alternatively rotor 202 may be a rigidcontainer having an off centered figure eight shaped chamber, which maycollect blood directly as discussed previously. Thus, while theembodiment described below illustrates a centrifuge having bag 226, itis to be understood that the alternative centrifuge bags disclosedherein may be used in a similar manner. The centrifuge 20 as discussedabove has an internal mid-shaft gear assembly 108 that provides themotive force to rotate the rotor assembly 200, and particularly therotor 202, at a rotation rate that is adequate to create centrifugalforces that act to separate the various constituents or components ofthe blood in the rotor 202. The drive assembly 822 may comprise a numberof devices useful for generating the motive force, such as an electricmotor with a drive shaft connected to internal drive components of thecentrifuge 20. In a preferred embodiment, the drive assembly 822comprises an electric motor that drives a belt attached to an exteriorportion of the centrifuge 20 and more particularly to the timing beltring 44. To obtain adequate separation, the rotation rate is typicallybetween about 0 RPM and 5000 RPM, and in one embodiment of theinvention, is maintained between about 0 RPM and 5000 RPM.

[0232] As discussed in detail previously, components of particulardensities assume radial positions or belts at differing distances fromthe central axis A of the rotor 202. For example, the heavier red bloodcells typically separate in an outer region while lower densityplatelets separate into a region more proximal to the central axis ofthe rotor 202. Between each of these component regions, there is aninterface at which the fluid density measurably changes from a higher toa lower density (i.e., as density is measured from an outer to an innerregion), and this density interface is used in some embodiments of thecentrifugal processing system 10 to identify the location of componentregions (as will be discussed in more detail below). In a preferredembodiment, the drive assembly 822 continues to operate to rotate thecentrifuge 20 to retain the separation of the components throughout theoperation of the centrifugal processing system 10.

[0233] Once blood separation has been achieved within the rotor 202, theoutlet pump 830 is operated to pump select components from the rotor 202through outlet lumen 828. As discussed previously, in relation to thefeatures of the disposable blood centrifuge bag 226, the centrifuge bagheld within the rotor 202 preferably is configured to allow theselective removal of a separated blood component, such as plateletslocated in a platelet rich plasma region, by the positioning of anoutlet lumen 232 a radial distance from the central axis of thecentrifuge bag 226. Preferably, this radial distance or radial locationfor the outlet lumen is selected to coincide with the radial location ofthe desired, separated component or the anticipated location of theseparated component. In this manner, the outlet pump 830 only (orsubstantially only) removes a particular component (such as plateletsinto container 400) existing at that radial distance. Once all or adesired quantity of the particular component is removed from thecentrifuge bag 226, operation of the outlet pump 830 is stopped, and anew separation process can be initiated. Alternatively, in a preferredembodiment, additional blood is pumped into the centrifuge by 226 byfurther operating the inlet pump 810 after or concurrent with operationof the outlet pump 830.

[0234] A concern with fixing the radial distance or location of theoutlet port is that each blood sample may have varying levels orquantities of different components. Thus, upon separation, the radialdistance or location of a particular component or component regionwithin the centrifuge bag 226 varies, at least slightly, with eachdifferent blood sample. Additionally, because of the varying levels ofcomponents, the size of the component region also varies and the amountthat can be pumped out of the centrifuge bag 226 by the outlet pump 830without inclusion of other components varies with each blood sample.Further, the position of the component region will vary in embodimentsof the separation system 10 in which additional blood is added after orduring the removal of blood by the outlet pump 830.

[0235] To address the varying location of a particular separatedcomponent, the centrifugal processing system 10 preferably is configuredto adjust the location of a separated component to substantially alignthe radial location of the separated component with the radial locationof the outlet port. For example, the centrifugal processing system 10may be utilized to collect platelets from a blood sample. In thisexample, the centrifugal processing system 10 preferably includes a redblood cell collector 812 connected to the inlet pump 810 via fluid line814 having an isolation valve 816 (e.g., a solenoid-operated valve orone-way check valve). Alternatively, the pump or syringe may also act asthe valve. The inlet pump 810 is configured to selectively pump fluidsin two directions, to and away from the centrifuge 820 through fluidline 818, and in this regard, may be a reversible-direction peristalticpump or other two-directional pump. Similarly, although shownschematically with two fluid lines 804 and 814, a single fluid line maybe utilized as an inlet and an outlet line to practice the invention.

[0236] Operation of the inlet pump 810 to remove fluid from thecentrifuge bag 226 is useful to align the radial location of the desiredseparated component with the outlet tube 250 and inlet tube 248 of thecentrifuge bag 226. When it is desired to align platelets or plateletrich plasma with the outlet tube 250, the inlet tube 248 connected tolumen 232 and 230, respectively, inlet tube 248 is preferably at agreater radial distance than the outlet tube 250. When suction isapplied to the inlet lumen 230 by inlet pump 810, red blood cells arepumped out of the centrifuge bag 226 and into the red blood cellcollector 812. As red blood cells are removed, the separated platelets(i.e., the desired component region) move radially outward to a newlocation within the centrifuge bag 226. The inlet pump 810 is operateduntil the radial distance of the separated platelets or platelet regionfrom the central axis is increased to coincide with the radial distanceor location of the outlet tube 250 of the centrifuge bag 226. Oncesubstantial alignment of the desired component region and the outlettube 250 is achieved, the outlet pump 830 is operated to remove all or aselect quantity of the components in the aligned component region.

[0237] To provide automation features of the invention, the centrifugalprocessing system 10 includes a controller 850 for monitoring andcontrolling operation of the inlet pump 810, the centrifuge 20, thedrive assembly 822, and the outlet pump 830. Numerous control devicesmay be utilized within the centrifugal processing system 10 toeffectively monitor and control automated operations. In one embodiment,the controller 850 comprises a computer with a central processing unit(CPU) with a digital signal processor, memory, an input/output (I/O)interface for receiving input and feedback signals and for transmittingcontrol signals, and software or programming applications for processinginput signals and generating control signals (with or without signalconditioners and/or amplifiers). The controller 850 is communicativelylinked to the devices of the centrifugal processing system 10 withsignal lines 860, 862, 864, 866, and 868 which may include signalconditioning devices and other devices to provide for propercommunications between the controller 850 and the components of thecentrifugal processing system 10.

[0238] Once blood is supplied to the blood source container 802, theoperator pushes the start button and the controller 850 transmits acontrol signal over signal line 864 to the drive assembly 822, which mayinclude a motor controller, to begin rotating the centrifuge 20 to causethe components of the blood in centrifuge bag 226 to separate intoradially-positioned regions (such as platelet rich plasma regions).After initiation of the centrifuge spinning or concurrently withoperation of the drive assembly 822, the controller 850 generates acontrol signal over signal line 860 to the inlet pump 810 to beginpumping blood from the blood source container 802 to the centrifuge bag226 of the centrifuge 20. In some embodiments of the processing system800, the drive assembly 822 is operable at more than one speed or over arange of speeds. Additionally, even with a single speed drive shaft therotation rate achieved at the centrifuge 20 may vary. To address thisissue, the processing system 10 may include a velocity detector 858 thatat least periodically detects movement of the centrifuge bag 226 portionof the centrifuge 20 and transmits a feedback signal over signal line866 to the controller 850. The controller 850 processes the receivedsignal to calculate the rotation rate of the centrifuge 20, and ifapplicable, transmits a control signal to the drive assembly 822 toincrease or decrease its operating speed to obtain a desired rotationrate at the centrifuge bag 226.

[0239] To determine when separation of the components in the centrifugebag 226 is achieved, the processing system 800 may be calibrated toaccount for variations in the centrifuge 20 and drive assembly 822configuration to determine a minimum rotation time to obtain a desiredlevel of component separation. In this embodiment, the controller 850preferably includes a timer mechanism 856 that operates to measure theperiod of time that the centrifuge 20 has been rotated by the driveassembly 822 (such as by beginning measuring from the transmission ofthe control signal by the controller 850 to the drive assembly 822).When the measured rotation time equals the calibrated rotation time fora particular centrifuge 20 and drive assembly 822 configuration, thetiming mechanism 856 informs the controller 850 that separation has beenachieved in the centrifuge bag 226. At this point, the controller 850operates to transmit control signal over signal line 860 to the inputpump 810 to cease operation and to the outlet pump 830 over signal line868 to initiate operation to pump a separated component in the componentregion adjacent the outlet port of lumen 232 of centrifuge bag 226through fluid line 828. In another embodiment where rotation time isutilized by controller 850, the velocity feedback signal from thevelocity detector 858 is utilized by the controller 850 to adjust therotation time as necessary to obtain the desired level of componentseparation. For example, the centrifugal processing system 10 can becalibrated for a number of rotation rates and the corresponding minimumrotation times can be stored in a look up table for retrieval by thecontroller 850 based on a calculated rotation rate. Rotational rates maybe varied either manually or automatically to optimize cellularcomponent position and or concentration.

[0240] Because the location of component separation regions variesduring separation operations, a preferred embodiment of the centrifugalprocessing system 800 includes a sensor assembly 840 to monitor theseparation of components within the centrifuge bag and to transmitfeedback signals over line 862 to the controller 850. As will beunderstood by those skilled in the art, numerous sensor devices existfor detecting the presence of certain components in a fluid, andspecifically a blood, sample. Many of these devices comprise a source ofradiant energy, such as infrared, laser, or incandescent light, and acompatible radiant energy-sensitive detector that reacts to the receivedenergy by generating an electric signal. Briefly, these radiant energydevices are useful because the detected signal varies in a measurablefashion with variances in the density of the material through whichbeams of the radiant energy are passed. According to the invention, thesensor assembly 840 may comprise any of these well-known types ofradiant energy source and detector devices and other sensor devicesuseful for measuring the existence of constituents of fluids such asblood.

[0241] The source and the detector of the sensor assembly 840 arepreferably located within the centrifugal processing system 800 to allowmonitoring of the centrifuge bag 226 and, particularly, to identify thepresence of a particular blood component in a radial position coincidingwith the radial position of the outlet port of the centrifuge bag 226.In one embodiment, the radiation beams from the source are transmittedthrough a “window” in the centrifuge bag 226 that has a radial locationthat at least partially overlaps the radial location of the outlet port.During operation of the centrifugal processing system 800, the feedbacksignals from the detector of the sensor assembly 840 allow thecontroller 850 to identify when a density interface has entered thewindow. This may occur for a number of reasons. When red blood cells arebeing removed by operation of the inlet pump 810 to remove fluid fromthe centrifuge bag 226 via the inlet tube 248. The change in density mayalso occur when a denser component is being added to the centrifuge bag226 causing the particular blood component to be pushed radially inward.In the centrifugation of whole blood, this occurs when additional bloodis added by operation of the input pump 810 and red blood cells collectin a region radially outward from the platelet region.

[0242] To account for differing movement of the density interface, thewindow of the radiation source may be alternatively positioned radiallyinward from the location of the outlet tube 250 of the centrifuge bag226. By positioning the window inward from the outlet tube 250, thecontroller 850 can identify when the outlet pump 830 has nearly removedall of the particular component of the monitored region and/or when theinlet pump 810 has removed a quantity of denser components causing themonitored region to move radially outward. The controller 850 can thenoperate to send control signals to turn off the outlet pump 830 or theinlet pump 810 (as appropriate) to minimize the amount of undesiredcomponents (lower density components) that enter the outlet tube 250.Alternatively, the sensor assembly 840 may have two radiation sourcesand detectors, and the second window of the sensor assembly 840 may belocated a distance radially outward from the outlet tube 250. With twosensing windows, the sensor assembly 840 is operable to provide thecontroller 850 information about a density interface moving radiallyinward toward the outlet tube 250 (such as when red blood cells areadded). In response, the controller 850 can generate a control signal tothe inlet pump 810 to operate to pump the denser components, such as redblood cells, out of the centrifuge bag 226. Two sensing windows alsoallow the controller 850 to detect a density interface moving outward,which allows the controller 850 to shut off the outlet pump 830 (and/orthe inlet pump 810 to stop evacuating processes) and/or to start theinlet pump 810 to add additional blood.

[0243] To further clarify operation of the processing system 800, FIG.54 is provided which illustrates the timing and relationship of controlsignals generated by the controller 850 and the receipt of feedbacksignals from the sensor assembly 840. In this embodiment, the radiationdetector of the sensor assembly 840 is positioned adjacent outlet tube(inlet to the outlet pump 830) in the centrifuge bag 226 to sensedensity changes in the fluid flowing past the outlet tube 250. Asillustrated, operation of the processing system 800 begins at time to,with the inlet pump 810, the outlet pump 830, and the centrifuge driveassembly 822 all being off or not operating. At time t₁, the controller850 operates in response to operator input or upon sensing the bloodsource 802 is adequately filled (sensor not shown) to generate a controlsignal on line 864 to begin operating the centrifuge drive assembly 822to rotate the centrifuge bag 226. In some embodiments, this controlsignal over line 864 also contains rotation rate information toinitially set the operating speed of the drive assembly 822.Concurrently or at a selected delay time, the controller 850 generates acontrol signal on line 860 to start the inlet pump 810 in aconfiguration to pump fluid to the centrifuge bag 226 over fluid line818. The sensor assembly 840 provides an initial density feedback signalto the controller 850 on line 862, which the controller 850 can processto determine an initial or unseparated density adjacent the outlet tube.Alternatively, the controller 850 may be configured to request afeedback signal from the sensor assembly 840 after a set delay period(as measured by the timer mechanism 856) to allow separation of thecomponents being pumped into the centrifuge bag 226 (such as thecalibrated, minimum rotation time discussed above) into regions.

[0244] At time t₂, the controller 850 functions to align the regionhaving the desired density, such as a region comprising a higher densityof platelets, adjacent the detector of the sensor assembly 840 (i.e.,adjacent the outlet tube). To achieve alignment, the controller 850transmits a control signal over line 860 to the inlet pump 810 to stoppumping fluid to the centrifuge bag 226, to reverse pumping directionsincluding shutting valve 806 and opening valve 816, and to begin pumpingcomponents having a higher density then the particular, desiredcomponent from the centrifuge bag 226 to the collector 812. For example,when the centrifugal processing system 10 is operated to separate andcollect platelets or platelet rich plasma, the inlet pump 810 at time,t₂, is operated to pump out the red blood cell fraction by applyingsuction at the inlet tube 248 to the centrifuge bag 226. At time t₃, thedensity of the fluid adjacent the outlet tube 250 begins to change asdenser components are removed by the inlet pump 810, and the sensorfeedback signal being transmitted to the controller 850 changes inmagnitude. The sensor feedback signal continues to change in magnitude(either becoming stronger or weaker depending on the particular sensorutilized and the material being collected) until at time t₄, when thecontroller 850 processes the feedback signal and determines that thedensity of the adjacent fluids is within a desired range. Thistransition can also be thought of as detecting when an interface betweentwo regions of differing densities passes by the location of thedetector of the sensor assembly 840.

[0245] With the region of the desired, separated component aligned withthe outlet tube 250, the controller 850 operates at time t₄, to send acontrol signal over line 860 to stop operations of the inlet pump 810.Also, at time t₄, or at any time thereafter, the controller 850generates a control signal over line 868 to begin operation the outletpump 830 to apply suction at the outlet tube 250 of the centrifuge bag226 to remove the desired component, such as the platelet rich plasmafraction, from the centrifuge bag 226. At time t₅, the sensor feedbacksignal again begins to change in magnitude as the density of the fluidnear the outlet tube 250 begins to change, such as when platelet poorplasma begins to enter the sampling window of the sensor assembly 840.At time t₆, the density of the fluid adjacent the outlet tube 250 and,hence, in the sampling window is outside of a desired density range(e.g., the fluid has less than a predetermined percentage of plateletsor other desired fluid component). In response, the controller 850transmits a control signal on line 868 to halt operations of the outletpump 830. Of course, the controller 850 can be operated to transmit thesignal to the outlet pump 830 at any time prior to time t₆, such as at atime after time t₅, when the density of the adjacent fluid begins tochange but prior to time t₆ or based on volume removed. The controller850 can then operate any time after time t₆, to halt operation of thecentrifuge drive assembly 822. Further, as discussed above, operationsof the separation centrifugal processing system 800 can be repeated withthe inlet pump 810 being operated to add additional fluid, e.g., blood,after time t₆. Alternatively, the inlet pump 810 and the outlet pump 830may be operated concurrently to add an additional volume of blood with acorresponding new amount of the component being collected after time t₄,to extend the period of time between detection of the interface at timet₄ and the detection of an out of range density at time t₆.

[0246] In the above discussion of the automated processing system 800, asensor assembly 840 was shown in FIG. 53 schematically, and it was notedthat the location of a radiant energy source and a detector may be anylocation within the processing system 800 useful for obtaining anaccurate measurement of separating blood components within thecentrifuge bag 226. For example, the source and detector can be bothpositioned within the centrifuge 20 at a location adjacent thecentrifuge bag 226. In this embodiment, problems may arise withproviding proper signal and power line connections to the source andsensor and with accounting for the rotation of the centrifuge andportions of the sensor assembly 840. Hence, one preferred embodiment ofthe processing system 800 provides for an externally positioned sensorassembly 840 including source and detector to simplify the structure ofthe centrifuge 20 while still providing effective density determinationsof fluids within the blood reservoir.

[0247]FIG. 55 illustrates a general side view of the relevant componentsof this external sensor embodiment of the centrifugal processing system800. Generally, the centrifuge 20 comprises a rotor extension portion880 and a drive portion 881, which is connected to the drive assembly822 (connection not shown). Both the centrifuge 20 and the rotorextension portion 880 rotate about a central or rotation axis, c_(axis),of the centrifuge 20. As discussed in more detail with respect to theinternal gearing features of the centrifuge 20, the drive portion 881spins in a ratio of 2 to 1 (or other suitable ratio) relative to thereservoir extension portion 880 to control twisting of inlet and outletfluid lines to the rotor extension portion 880. The internal gearingfeatures of the centrifuge 20 also enable the centrifuge 20 toeffectively obtain rotation rates that force the separation ofcomponents with differing densities while limiting the risk that densercomponents, such as red blood cells, will become too tightly packedduring separation forming a solid, dense material that is more difficultto pump or remove from the centrifuge 20.

[0248] Referring again to FIG. 55, the rotor extension portion 880 isshown located on the upper end of the centrifuge 20 and includes acentrifuge bag 226 or other receptacle. Preferably, the rotor extensionportion 880 is fabricated from a transparent or partially transparentmaterial, such as any of a number of plastics, to allow sensing of fluiddensities. The rotor extension portion 880 extends a distance, d_(over),beyond the outer edge of the centrifuge 20 as measured radially outwardfrom the central axis, c_(axis). The distance, d_(over), is preferablyselected such that the desired component, such as the platelet richplasma fraction, to be collected readily separates into a region at apoint within the centrifuge bag 226 that also extends outward from thecentrifuge 20. In this regard, the rotor extension portion 880 is alsoconfigured so that the centrifuge bag 226 extends within the rotorextension portion 880 to a point near the outer circumference of therotor extension portion 880. The distance, d_(over), selected forextending the rotor extension portion 880 is preferably selected tofacilitate alignment process (discussed above) and to control the needfor operating the input pump 810 to remove denser components. In oneembodiment, the distance, d_(over), is selected such that duringseparation of a typical blood sample center of the platelet rich regionis about one half the extension distance, d_(over), from thecircumferential edge of the centrifuge 20.

[0249] The sensor assembly 840 is entirely external to the centrifuge 20as shown in FIG. 55. The sensor assembly 840 includes a source 882 foremitting beams 884 of radiant energy into and through the rotorextension portion 880 and the included centrifuge bag 226. Again, asdiscussed previously, the radiant energy source 882 may be nearly anysource of radiant energy (such as incandescent light, a strobe light, aninfrared light, laser and the like) useful in a fluid density sensor andthe particular type of detector or energy used is not as important asthe external location of the source 882. The sensor assembly 840 furtherincludes a detector 886 that receives or senses beams 888 that havepassed through the centrifuge bag 226 and have impinged upon thedetector 886. The detector 886 is selected to be compatible with thesource 882 and to transmit a feedback signal in response sensing theenergy beams 888. The detector 886 (in combination with the controller850 and its processing capacities) is useful for detecting the densityof fluids in the centrifuge bag 226 between the source 882 and thedetector 886. Particularly, the sensor assembly 840 is useful foridentifying changes in fluid density and interfaces between fluids withdiffering densities. For example, the interface between a regioncontaining separated red blood cells and a region containing theplatelet rich plasma fraction, and the interface between the plateletrich plasma region and a platelet-poor plasma region.

[0250] With some source and detector configurations, a sampling windowis created rather than a single sampling point (although a singlesampling point configuration is useful as part of the invention ascreating a window defined by a single radial distance). The samplingwindow is defined by an outer radial distance, d_(OUT), from the centralaxis, c_(axis), and an inner radial distance, d_(IN). As may beappreciated, for many source and detector configurations the size of thesampling window may be rather small approximating a point and may, ofcourse vary in cross-sectional shape (e.g., circular, square,rectangular, and the like). As discussed previously, it is preferablethat the sensor assembly 840 be positioned relative to the reservoirextension portion 880 and the centrifuge bag 226 such that the samplingwindow created by the source 882 and detector 886 at least partiallyoverlaps the radial position of the region created during separationprocesses containing a component of particular density, such asplatelets. This may be a calibrated position determined throughcalibration processes of the centrifuge 20 in which a number of blood(or other fluid) samples are fully separated and radial distances to aparticular region are measured. The determined or calibrated positioncan then be utilized as a initial, fixed location for the sensorassembly 840 with the source 882 and detector 886 being positionedrelative to the rotor extension portion 880 such that the samplingwindow overlaps the anticipated position of the selected separationregion. Of course, each sample may vary in content of various componentswhich may cause this initial alignment to be inaccurate and operationsof the centrifugal processing system 800 may cause misalignment ormovement of regions. Hence, alignment processes discussed abovepreferably are utilized in addition to the initial positioning of thesampling window created by the sensor assembly 840.

[0251] In an alternate embodiment, the sensor assembly 840 is not in afixed position within the separation system 800 and can be positionedduring separation operations. For example, the sensor assembly 840 maybe mounted on a base which can be slid radially inward toward thecentrifuge 20 and radially outward away from the centrifuge 20 to varythe distances, d_(IN) and d_(OUT). This sliding movement is useful forproviding access to the centrifuge bag 226, such as to insert and removea disposable bag. During operation, the sensor assembly 840 wouldinitially be pushed outward from the centrifuge 20 until a new bag wasinserted into the centrifuge bag 226. The sensor assembly 840 could thenbe slid inward (or otherwise moved inward) to a calibrated position.Alternatively, the centrifugal processing system 800 could be operatedfor a period of time to achieve partial or full separation (based on atimed period or simple visual observation) and then the sensor assembly840 slid inward to a position that the operator of the centrifugalprocessing system 800 visually approximates as aligning the samplingwindow with a desired region of separated components (such as theplatelet rich plasma region). The effectiveness of such alignment couldthen readily be verified by operating the sensor assembly 840 to detectthe density of the fluids in the centrifuge bag 226 and a calculateddensity (or other information) could be output or displayed by thecontroller 850. This alternate embodiment provides a readilymaintainable centrifugal processing system 800 while providing thebenefits of a fixed position sensor assembly 840 and added benefits ofallowing easy relative positioning to obtain or at least approximate adesired sample window and separation region alignment.

[0252] In some situations, it may be preferable to not have a rotorextension portion 880 or to modify the rotor extension portion 880 andthe sensor assembly 840 such that the extension is not significant tomonitoring the separation within the blood reservoir or centrifuge bag226. Two alternative embodiments or arrangements are illustrated inFIGS. 56 and 57 that provide the advantages of an external sensorassembly 840 (such as an external radiation source and detector). Withthese further embodiments provided, numerous other expansions of thediscussed use of an external sensor will become apparent to thoseskilled in the arts and are considered within the breadth of thisinvention.

[0253] Referring to FIG. 56, a rotor 202 is illustrated that has noextending portion (although some extension may be utilized) and containsthe centrifuge bag 226. Again, the rotor 202 and centrifuge bag 226 arepreferably fabricated from plastics or other materials that allowradiation to pass through to detect changes in densities or otherproperties of fluid samples within the centrifuge bag 226. In thisembodiment of the sensor assembly 840, the radiation source 882 and thedetector 886 are not positioned on opposing sides of the rotor 202.Instead, a reflector 885 (such as a mirror and the like) is positionedwithin the drive portion 881 of the centrifuge to receive the radiationbeams 884 from the radiation source 882 and direct them through theportion 880 and centrifuge bag 226. The detector 886 is positionedwithin the sensor assembly 840 and relative to the centrifuge 20 toreceive the deflected or reflected beams 888 that have passed throughthe fluid sample in the centrifuge bag 226. In this manner, the samplingwindow within the centrifuge bag 226 can be selected to align with theanticipated location of the fraction that is to be collected uponseparation. In a preferred embodiment, the sampling window at leastpartially overlaps with the location of the outlet tube of the bloodreservoir or centrifuge bag 226.

[0254] In one embodiment, the drive portion is fabricated from anon-transparent material and a path for the beams 884 from the radiationsource 884 to the reflector 885 is provided. The path in one preferredembodiment is an opening or hole such as port 154 or 156 (FIG. 14) inthe side of the drive portion 881 that creates a path or tunnel throughwhich the beams 884 travel unimpeded. Of course, the opening may bereplaced with a path of transparent material to allow the beams totravel to the reflector 885 while also providing a protective cover forthe internals of the drive portion 881. A path is also provideddownstream of the reflector 885 to allow the beams 884 to travel throughthe drive portion 881 internals without or with minimal degradation.Again, the path may be an opening or tunnel through the drive portionleading to the portion 202 or be a path created with transparentmaterials. The beams 884 in these tunnel path embodiments enter thedrive portion 881 one time per revolution of the drive portion 881,which provides an acceptable rate of sampling. Alternatively, areflector 885 may readily be provided that extends circumferentiallyabout the center axis of the drive portion 881 to provide a samplingrate equivalent to the rate of beam 884 transmission. Of course, thepositions of the radiation source 882 and the detector 886 may bereversed and the angle of the reflector 885 and transmission of thebeams 884 may be altered from those shown to practice the invention.

[0255] A further embodiment of an external sensor assembly 840 isprovided in FIG. 57. In this embodiment, the radiation source 882 alsoacts as a radiation detector so there is no need for a separatedetector. In this more compact external sensor configuration, theradiation source and detector 882 transmits beams 884 into the rotatingdrive portion 881 through or over the path in the drive portion 881. Thereflector 885 reflects the beams 884 toward the rotor 202 and thecentrifuge bag 226 to create a sampling window within the centrifuge bag226 in which density changes may be monitored. After passing through thecentrifuge bag 226 and included fluid sample, the beams 888 strike asecond reflector 887 that is positioned within the rotor 202 to reflectthe beams 888 back over the same or substantially the same path throughthe centrifuge bag 226 to again strike the reflector 885. The reflector885 directs the beams 888 out of the drive portion 881 and back to theradiation source and detector 882 which, in response to the impingingbeams 888, transmits a feedback signal to the controller 850 for furtherprocessing.

[0256] In one embodiment, the beams 884 enter the driving portion 881once during every revolution of the driving portion 881. The portion 880is preferably rotating twice for every rotation of the driving portion881, as discussed in detail above, and hence, the second reflector 887is aligned to receive the beams 888 only on every other rotation of thedriving portion 881. Alternatively, a pair of reflectors 887 may bepositioned in the rotor 202 such that the beams 888 may be received andreflected back through the centrifuge bag 226 once for every rotation ofthe driving portion 881. In yet a further embodiment, the reflector 885and second reflector 887 may expand partially or fully about the centeraxis of the centrifuge 20 (with corresponding openings and/ortransparent paths in the driving portion 881) to provide a highersampling rate.

[0257] According to an important feature of the invention, temperaturecontrol features are provided in an alternate embodiment of theautomated processing system invention 900, as illustrated in FIG. 58.Providing temperature controls within the processing system 900 can takemany forms such as controlling the temperature of input fluid samplesfrom the blood source 802, monitoring and controlling the temperature offluids in the centrifuge bag 226 to facilitate separation processes, andcontrolling the operating temperature of temperature sensitivecomponents of the processing system 900. These components include butare not limited to, red blood cells, white blood cells, plasma, plateletrich plasma or any of these components mixed with other drugs, proteinsor compounds. In a preferred embodiment of the invention, a temperaturecontrol system is included in the processing system 900 to heatcomponents removed from the centrifuge bag 226 by the outlet pump 830 toa desired temperature range. For example, when the processing system 900is utilized in the creation of autologous platelet gel, a dispenserassembly 902 is included in the processing system 900 and includeschambers or syringes for collecting and processing platelet rich plasmadrawn from the centrifuge 20. As part of the gel creation process, it istypically desirable to activate the platelets in the harvested plateletrich plasma fraction prior to the use of the gel (e.g., delivery to apatient). The temperature control system is useful in this regard forraising, and for then maintaining, the temperature of the platelets inthe dispenser assembly to a predetermined activation temperature range.In one embodiment of the gel creation process, the activationtemperature range is 25° C. to 50° C. and preferably 37° C. to 40° C.,but it will be understood that differing temperature ranges may readilybe utilized to practice the invention depending on the desiredactivation levels and particular products being processed or createdwith the processing system 900.

[0258] Referring to FIG. 58, the temperature control system of theprocessing system 900 includes a temperature controller 904 that iscommunicatively linked to the controller 850 with feedback signal line906. The controller 850 may be utilized to initially set operatingtemperature ranges (e.g., an activation temperature range) andcommunicate these settings over feedback signal line 906 to thetemperature controller 904. Alternatively, the temperature controller904 may include input/output (I/O) devices for accepting the operatingtemperature ranges from an operator or these ranges may be preset aspart of the initial fabrication and assembly of the processing system900. The temperature controller 904 may comprise an electronic controlcircuit allowing linear, proportional, or other control overtemperatures and heater elements and the like. In a preferredembodiment, the temperature controller 904 includes a microprocessor forcalculating sensed temperatures, memory for storing temperature andcontrol algorithms and programs, and I/O portions for receiving feedbacksignals from thermo sensors and for generating and transmitting controlsignals to various temperature control devices (e.g., resistive heatelements, fan rotors, and other devices well-known to those skilled inthe heating and cooling arts).

[0259] As illustrated, a temperature sensor 908 comprising one or moretemperature sensing elements is provided to sense the temperature of thedispenser assembly 902 and to provide a corresponding temperaturefeedback signal to the temperature controller 904 over signal line 910(such as an electric signal proportional to sensed temperature changes).The temperature sensor 908 may be any temperature sensitive deviceuseful for sensing temperature and, in response, generating a feedbacksignal useful by the temperature controller 904, such as a thermistor,thermocouple, and the like. In a preferred embodiment, the temperaturesensor 908 is positioned within the dispenser assembly 902 to be in heattransferring or heat sensing contact with the syringes or other chamberscontaining the separated product which is to be activated. In thismanner, the temperature controller 904 is able to better monitor whetherthe temperature of the relevant chambers within the dispenser assembly902 is within the desired activation temperature range.

[0260] To maintain the chambers of the dispenser assembly 902 within atemperature range, a heater element 913 is included in the temperaturecontrol system and is selectively operable by the temperature controller904 such as by operation of a power source based on signals receivedfrom the temperature sensor 908. The heater element 913 may comprise anynumber of devices useful for heating an object such as the chambers ofthe dispenser assembly 902, such as a fluid heat exchanger with tubingin heat exchange contact with the chambers. In a preferred example, butnot as a limitation, electrical resistance-type heaters comprisingcoils, plates, and the like are utilized as part of the heater element913. Preferably, in this embodiment, the resistive portions of theheater element 913 would be formed into a shape that conforms to theshape of the exterior portion of the chambers of the dispenser assembly902 to provide efficient heat transfer but preferably also allow forinsertion and removal of the chambers of the dispenser assembly 902.During operation of the separation system 900, the temperaturecontroller 904 is configured to receive an operating temperature range,to receive and process temperature feedback signals from the temperaturesensor 908, and in response, to selectively operate the heater element913 to first raise the temperature of the chambers of the dispenserassembly 902 to a temperature within the operating temperature range andto second maintain the sensed temperature within the operating range.

[0261] For example, a desired operating range for activating a gel ormanipulating other cellular components and their reactions ontothemselves or with agents may be provided as a set point temperature (ordesired activation temperature) with a tolerance provided on either sideof this set point temperature. The temperature controller 904, in thisexample, may operate the heater element 913 to raise the temperature ofthe chambers of the dispenser assembly 902 to a temperature above theset point temperature but below the upper tolerance temperature at whichpoint the heater element 913 may be shut off by the temperaturecontroller 904. When the temperature sensed by the temperature sensor908 drops below the set point temperature but above the lower tolerancetemperature, the temperature controller 904 operates the heater element913 to again raise the sensed temperature to above the set pointtemperature but below the upper tolerance temperature. In this manner,the temperature controller 904 effectively maintains the temperature ofthe chambers in the dispenser assembly 902 within a desired activationtemperature range (which, of course, may be a very small range thatapproximates a single set temperature). In one embodiment, thetemperature controller is or operates as a proportional integralderivative (PID) temperature controller to provide enhanced temperaturecontrol with smaller peaks and abrupt changes in the temperatureproduced by the heater element 913. Additionally, the temperaturecontroller 904 may include visual indicators (such as LEDs) to indicatewhen the sensed temperature is within a set operating range and/or audioalarms to indicate when the sensed temperature is outside the setoperating range.

[0262] In another embodiment, the heater element 913 is configured tooperate at more than one setting such that it may be operated throughoutoperation of the processing system 900 and is not shut off. For example,the heater element 913 may have a lower setting designed to maintain thechambers of the dispenser assembly 902 at the lower end of the operatingrange (e.g., acceptable activation temperature range) with highersettings that provide heating that brings the chambers up to highertemperatures within the set operating range. In another embodiment, theheater element 913 is configured to heat up at selectable rates (e.g.,change in temperature per unit of time) to enhance the activation orother processing of separated liquids in the dispenser assembly 902.This feature provides the temperature controller 904 with control overthe heating rate provided by the heater element 913.

[0263] As discussed previously, the invention provides features thatcombine to provide a compact separation system that is particularlyadapted for onsite or field use in hospitals and similar environmentswhere space is limited. FIG. 59 illustrates one preferred arrangement ofthe centrifugal processing system 900 of FIG. 58 that provides a compactprofile or footprint while facilitating the inclusion of a temperaturecontrol system. An enclosure 916 is included as part of the temperaturecontrol system to provide structural support and protection for thecomponents of the temperature control system. The enclosure 916 may befabricated from a number of structural materials, such as plastic. Theenclosure 916 supports a heater housing 918 that is configured to allowinsertion and removal of the chambers and other elements of thedispenser assembly 902. The heater housing 918 has a wall that containsthe heater element 913 (not shown in FIG. 59) which is connected viacontrol line 914 to the temperature controller 904. The temperaturesensor 908 (not shown in FIG. 59) is also positioned within the heaterhousing 918, and as discussed with reference to FIG. 58, is positionedrelative to the chambers of the dispenser assembly 902 to sense thetemperature of the chambers, and the contained fluid, during operationof the system 900. A temperature feedback signal is transmitted by thetemperature sensor 908 over line 910 to temperature controller 904,which responds by selectively operating the heater element 913 tomaintain the temperature within the heater housing 918 within a selectedoperating range.

[0264] Because the separation system 900 includes temperature sensitivecomponents, such as the controller 850, the temperature control systempreferably is configured to monitor and control the temperature withinthe enclosure 916. As illustrated, a temperature sensor 920 is includedto sense the ambient temperature within the enclosure 916 and totransmit a feedback signal over line 922 to temperature controller 904.An air inlet 930, such as a louver, is provided in the enclosure 916 toallow air, A_(IN), to be drawn into and through the enclosure 916 toremove heated air and maintain the temperature within the enclosure 916at an acceptable ambient temperature. To circulate the cooling air, afan 934 is provided to pull the air, A_(IN), into the enclosure 916 andto discharge hotter air, A_(OUT), out of the enclosure 916. The fan 934is selectively operable by the temperature controller 904 via controlsignals over line 938. The size or rating of the fan 934 may vary inembodiments of the invention and is preferably selected based on thevolume of the enclosure 916, the components positioned within theenclosure 916 (e.g., the quantity of heat generated by the separationsystem 900 components), the desired ambient temperature for theenclosure 916, and other cooling design factors.

[0265] In an alternate embodiment of the present invention a dispenser902, as shown in FIG. 60, is provided, for manipulating the cellularfraction which has been isolated and collected via outlet lumen 232. Ingeneral, the present invention relates to a dispenser 902 which allowsfor a manual or automated manipulation of a two-phase method for formingan autologous platelet gel 970 composition wherein all of the bloodcomponents for the platelet gel 970 are derived from a patient to whomthe platelet gel 970 will be applied.

[0266] The methods of the present invention for preparing an autologousplatelet gel 970 composition, discussed in further detail below, arerepresented in the flow diagrams depicted in FIGS. 61-63. As discussedpreviously, the methods of the present invention begins by forminganticoagulated whole blood 396 which is achieved by collecting apatient's whole blood 394 in a source container 398 having ananticoagulation agent, such as sodium citrate (citrate) or heparin.Preferably, the whole blood 394 is collected and mixed with a 3.8%solution of sodium citrate (referred to herein as “citrate collectionmedium”) specifically in a 9:1 ratio of blood to citrate collectionmedium. A 3.8% solution of sodium citrate is prepared by adding 3.8grams of sodium citrate per 100 ml of water. While a 3.8% sodium citratecollection medium is that which is frequently used to collect andpreserve blood, the person skilled in this art will recognize that theratio of sodium citrate to whole blood could be in the range of about10.9-12.9% MMOL, final concentration.

[0267] First, as discussed in detail previously and depicted in FIG. 61,platelet rich plasma 260 and/or platelet poor plasma 262 are formed bycentrifuging a quantity of anticoagulated whole blood 396 that waspreviously drawn from the patient. The platelet rich plasma 260 is firstdrawn from the centrifuge bag 226 and into collection chamber 400.Collection chamber 400 is preferably a syringe, but any container thatwill not contact activate the collected fraction is acceptable. Theplatelet rich plasma 260 can be pumped via outlet pump 830 (FIG. 53)into a collection chamber 400 or the desired fraction can be drawndirectly into dispenser 902.

[0268] In the preferred embodiment, depicted in FIG. 62 according toroute 951, the platelet rich plasma 260, in centrifuge bag 226, isdivided into two portions and stored in vessels 952 and 960. The firstportion is approximately ¼ to ½ of the total volume of platelet richplasma 260 and is utilized in phase-one to prepare the thrombin, whilethe second portion of platelet rich plasma 260 is utilized in phase-twovessel 960. Once the platelet rich plasma 260 or alternatively theplatelet poor plasma 262 (shown in FIG. 61) is obtained, the preferredmethods to obtain thrombin and then produce the platelet gelcompositions in an expedited manner, that is, in less than threeminutes, are detailed diagrammatically in routes 951 or 981, shown inFIGS. 62 and 63, respectively and discussed in detail below. If,however, a longer clotting time, that is, in a range of two to eightminutes, is desirous the method to obtain the platelet gel compositionof the present invention can proceed along the routes 971 and 987, whichare also detailed diagrammatically in FIGS. 63 and 63, respectively anddiscussed in detail below.

[0269] Phase-one according to the preferred embodiment (FIG. 62) beginsby restoring the clot-forming process. To accomplish this, an agent(restoration agent) capable of reversing the effects of theanticoagulation agent is added back into the first portion of theplatelet rich plasma 260 stored in vessel 952. Preferably, therestoration agent can be vessel 952 itself or the restoration agent iscontained within vessel 952 prior to the introduction of platelet richplasma 260; however, the restoration agent may also be introduced later.It is furthermore preferable that the contact activator be a materialsuch as but not limited to glass wool 953 or silica, aluminum,diatomaceous earth, kaolin, etc., or non-wettable surfaces such asplastic, siliconized glass, etc. Chemical activators, such as kaolin,can also be used to speed up the clotting time; however, theirsubsequent removal would also be necessary. In the preferred embodiment,a plastic syringe is the preferred container used to collect the desiredfraction. In the presently preferred embodiment of the invention, thereversal of the anticoagulant is accomplished using calcium chloride.However, any substance which is known or found to be functionallyequivalent to calcium chloride, such as, calcium gluconate or calciumcarbonate, in restoring the coagulation activity of citrated blood maybe used in the practice of the present invention. Thus, although calciumchloride is the presently preferred calcium salt for use in theinvention, any calcium salt which functions in a similar manner tocalcium chloride may be used in the invention. Similarly, although manyblood coagulation reactions are currently believed to require calciumions as cofactors, any substance which is known or subsequently found tobe functionally equivalent to calcium in facilitating these coagulationreactions may be used, either individually or in combination withcalcium, in the practice of the present invention. If theanticoagulation agent used was heparin, then heparinase or any othersuitable anticoagulant reversing compound would be used to reverse theeffect of the anticoagulation agent. The concentration of therestoration agent used to reverse the anticoagulation will depend inpart, upon the concentration of the anticoagulation agent in theplatelet rich plasma 260 and the stoichiometry of the chelating andcoagulation reactions. However, the concentration of the restorationagent used to reverse the anticoagulation must be sufficient to achieveclot formation.

[0270] Upon restoration of the platelet rich plasma 260 as shown inFIGS. 62, a clot 954 will naturally form. The resulting clot 954 is thentriturated by squeezing the clot 954 through glass wool 953 which servesnot only as a contact activator but also as a filter, thus expressingthrombin 955. Alternatively, or in addition a filter 958 having a largemicron pore size thereby allowing the removal of clot debris and anyactivator or solids that are present. Filter 958 is positioned at theoutlet 956 of vessel 952. In the preferred embodiment, the thrombin 955is then mixed with the second portion of platelet rich plasma (PRP) 260contained within vessel 960 to form the platelet gel composition 970 ofthe present invention in less than three minutes and in quantitiessufficient for clinical use.

[0271] Other additives can be added to the above-described process toincrease the concentration of thrombin formed by the intrinsic pathwayor the extrinsic pathway.

[0272] As discussed in detail above, restoring the clotting cascadefunction of citrated plasma by addition of calcium chloride and exposureto an activating agent such as glass wool can generate autologousthrombin. The yield of autologous thrombin by this method however, maybe low due to incomplete conversion of prothrombin and the inactivationof generated thrombin by fibrin and antithrombin III. The addition ofmodifying agents, such as epsilon aminocaproic acid, to the plasma mayimprove the yield by reducing the amount of thrombin neutralization. Thegreatest improvement in thrombin yield, however, will be achieved byproviding a thromboplastic material upon which the necessary clottingfactors will assemble to maximize the rate of prothrombin conversion.The activated platelet membrane provides such a stimulant surface andalso enriches the necessary factor V activity by secreting additionalfactor V during platelet degranulation. The addition of exogenouslipoprotein and/or thromboplastic material to the plasma environment mayalso serve to maximize the thrombin generation by activation of bothintrinsic and extrinsic clotting cascades. Additional amplification ofautologous thrombin generation may also be attained by pretreatment ofPRP and/or PPP to block or remove both antithrombin-III and fibrinogenprior to conversion of prothrombin to thrombin. Such modification may beattained by use of appropriate adsorptive agents, antibodies orprecipitating reagents.

[0273] In an alternative embodiment, thrombin 950 is mixed with theplatelet poor plasma 262 of phase-two thereby forming the autologousplatelet gel composition 972 of the present invention in less than threeminutes.

[0274] A third embodiment of the present invention, route 971, shown inFIG. 62, contemplates collecting the original quantity of platelet richplasma (PRP) 260 derived from the anticoagulated whole blood 396 in acontainer, having a wettable surface, such as glass. The platelet richplasma 260 is then recalcified and the platelet gel composition 974forms. The desired platelet gel composition 974 will requireapproximately two to eight minutes to form as opposed to less than athree minute formation as was described in the preferred embodiment.

[0275] In the fourth embodiment depicted diagrammatically by route 981in FIG. 63, the platelet poor plasma 262, rather then the platelet richplasma 260, is divided into two portions, as discussed previously in thepreferred embodiment. The first portion, used in phase-one, which isapproximately ¼ to ½ the original volume is stored in a vessel 952having a wettable surface, then the restoration agent, preferablycalcium chloride, is added directly to the platelet poor plasma 262.Surface activation of the restored platelet poor plasma 262 occurs asresult of the vessel's surface and/or the glass wool 953 or othersurface or chemical activators and a clot 962 thus forms. The resultingclot 962 is triturated, as described previously, and the thrombin 955 iscollected. Thrombin 955 is then mixed with the platelet rich plasma 260of phase-two thereby forming the platelet gel sealant composition 973.

[0276] In the fifth embodiment, thrombin 955 is mixed with the plateletpoor plasma 262 of phase-two thereby forming the platelet gelcomposition 975 in less than three minutes.

[0277] The sixth embodiment follows route 987, shown in FIG. 63 whereinthe original quantity of platelet poor plasma 262 is collected in acontainer having a wettable surface, such as glass. The platelet poorplasma 262 is then recalcified and the platelet gel composition forms.

[0278] The tensile strength of the platelet gel compositions of thepresent invention can be effected by the addition of calcium ions.Consequently, if a stronger bioadhesive sealant composition is desiredusing the methods discussed above and disclosed in routes 951 and 981,in FIGS. 62 and 62, respectively, more calcium ions may be added at thetime the serum is introduced into the platelet rich plasma 260 or theplatelet poor plasma 262. Alternatively, if the method of preparing theplatelet gel compositions follows routes 971 and 987, depicted in FIGS.62 and 63, respectively, then calcium ions may be introduced directlyinto the platelet rich plasma 260 or the platelet poor plasma 262 andthe platelet gel compositions 974 and 976, respectively, will form.

[0279] As discussed in further detail below, the time period necessaryfor the formation of the platelet gel composition of the presentinvention is dependent on the quantity of serum added. A 1:4, 1:2 and3:4 ratio of serum to platelet rich plasma or platelet poor plasmaresults in the formation of the platelet gel composition inapproximately 90, 55 and 30 seconds, respectively. Furthermore, due tothe fact that thrombin is autocatalytic, it is important that the serumbe used within five hours of preparation, preferably within two hoursand ideally immediately. Alternatively, the serum can be chilled orfrozen indefinitely.

[0280] The platelet gel compositions of this invention may be used forsealing a surgical wound by applying to the wound a suitable amountplatelet rich plasma or platelet poor plasma. Moreover, due to the factthat the platelet gel compositions of the present invention have beenprepared solely from blood components derived from the patient that isto receive the platelet gel there is a zero probability of introducing anew blood transmitted disease to the patient. The methods of the presentinvention may be further modified so that the formed platelet gelcomposition functions not only as a haemostatic agent, but also as anadjunct to wound healing and as a matrix for delivery of drugs andproteins with other biologic activities. For example, it is well knownthat fibrin glue has a great affinity to bind bone fragments which isuseful in bone reconstruction, as in plastic surgery or the repair ofmajor bone breaks. Consequently, in keeping with the autologous natureof the platelet gel composition of the present invention autologous bonefrom a patient can be ground or made into powder or the like, and mixedinto the platelet rich plasma obtained in phase-two of the methods ofthe present invention. Autologous thrombin is then mixed in with theplatelet rich plasma and bone fragments in an amount sufficient to allowthe resulting gel to be applied to the desired locale where it congeals.Other materials that may be utilized are, but not limited to, gelatincollagen, degradable polymers, hyaluronic acid, carbohydrates andstarches.

[0281] In instances where the desired platelet gel composition of thepresent invention is to further function as a delivery device of drugsand proteins with other biologic activities the method of the presentinvention may be modified as follows. Prior to adding the thrombin,obtained in phase-one, to the platelet rich plasma of phase-two a widevariety of drugs and proteins with other biologic activities may beadded to the platelet rich plasma of phase-two. Examples of the agentsto be added to the platelet rich plasma prior to the addition of theserum include, but are not limited to, analgesic compounds, such asLidocaine, antibacterial compounds, including bactericidal andbacteriostatic compounds, antibiotics (e.g., adriamycin, erythromycin,gentimycin, penicillin, tobramycin), antifungal compounds,anti-inflammatories, antiparasitic compounds, antiviral compounds,anticancer compounds, such as paclitaxol enzymes, enzyme inhibitors,glycoproteins, growth factors (e.g. lymphokines, cytokines), hormones,steroids, glucocorticosteroids, immunomodulators, immunoglobulins,minerals, neuroleptics, proteins, peptides, lipoproteins, tumoricidalcompounds, tumorstatic compounds, toxins and vitamins (e.g., Vitamin A,Vitamin E, Vitamin B, Vitamin C, Vitamin D, or derivatives thereof). Itis also envisioned that selected fragments, portions, derivatives, oranalogues of some or all of the above may be used.

[0282] A number of different medical apparatuses and testing methodsexist for measuring and determining coagulation and coagulation-relatedactivities of blood. These apparatuses and methods can be used to assistin determining the optimal formulation of activator, that is, thrombin,calcium and plasma necessary to form the platelet gel composition of thepresent invention. Some of the more successful techniques of evaluatingblood clotting and coagulation are the plunger techniques illustrated byU.S. Pat. No. 4,599,219 to Cooper et al., U.S. Pat. No. 4,752,449 toJackson et al., and U.S. Pat. No. 5,174,961 to Smith, all of which areassigned to the assignee of the present invention, and all of which areincorporated herein by reference.

[0283] Automated apparatuses employing the plunger technique formeasuring and detecting coagulation and coagulation-related activitiesgenerally comprise a plunger sensor cartridge or cartridges and amicroprocessor controlled apparatus into which the cartridge isinserted. The apparatus acts upon the cartridge and the blood sampleplaced therein to induce and detect the coagulation-related event. Thecartridge includes a plurality of test cells, each of which is definedby a tube-like member having an upper reaction chamber where a plungerassembly is located and where the analytical test is carried out, and areagent chamber which contains a reagent or reagents. For an activatedclotting time (ACT) test, for example, the reagents include anactivation reagent to activate coagulation of the blood. A plug memberseals the bottom of a reagent chamber. When the test commences, thecontents of the reagent chamber are forced into the reaction chamber tobe mixed with the sample of fluid, usually human blood or itscomponents. An actuator, which is a part of the apparatus, lifts theplunger assembly and lowers it, thereby reciprocating the plungerassembly through the pool of fluid in the reaction chamber. The plungerassembly descends by the force of gravity, resisted by a property of thefluid in the reaction chamber, such as its viscosity. When the propertyof the sample changes in a predetermined manner as a result of the onsetor occurrence of a coagulation-related activity, the descent rate of theplunger assembly there through is changed. Upon a sufficient change inthe descent rate, the coagulation-related activity is detected andindicated by the apparatus.

[0284] Using the methods discussed above, cartridges were assembled withserum obtained from either platelet rich plasma or platelet poor plasma,and CaCl₂ in the reagent chambers. Clotting time tests were performed bythe automated process with either platelet rich plasma (PRP) or plateletpoor plasma (PPP) dispersed into the reaction chambers of thecartridges. In the first experiment, the results of which arerepresented in FIG. 64, the amount of serum, the type of plasma fromwhich the serum was derived, and the type of plasma the serum was mixedwith were tested to determine the shortest clotting times. The ratios ofserum to platelet rich plasma or platelet poor plasma that were studiedincluded 1:4, 1:2, and 3:4. In the second set of experiments, theresults of which are represented in FIGS. 66 and 67, the relationshipbetween actual gel time for the platelet gel of the present was comparedto the clotting time in the cartridge, wherein there is a 0, 30, or 60minute delay of adding the serum from its generation. The third set ofexperiments, the results of which are represented in FIGS. 68 and 69,studied the effect of calcium addition on actual gel time versusclotting time in the cartridge. The final set of experiments, theresults of which are represented in FIG. 65, studied the effect ofadding calcium on clotting times.

[0285] Although clotting times varied among donors, comparisons ofclotting times for individual donors show significant effects of theserum to plasma ratio and the calcium concentration. For all donors, theshortest clotting times occurred for the 3:4 ratio, with clotting timesthat were 47% shorter than those for the 1:4 ratio. Although thedifference in clotting times for the 3:4 ratio and the 1:2 ratio was notstatistically significant, the clotting times were consistently shorterusing the 3:4 ratio for all donors. These results demonstrate thatclotting times may be shortened by increasing the serum to platelet richplasma ratio. Similarly, clotting times were significantly affected bythe amount of calcium added, with the shortest clotting times obtainedwhen no calcium was added, suggesting that the serum contained levels,of calcium that were sufficient to recalcify the citrated platelet richplasma. Preliminary results from the scale-up experiments suggest thatexperimental clotting times in the cartridges correlate with actual geltimes.

[0286] The invention is further illustrated by the following non-limitedexamples. All scientific and technical terms have the meanings asunderstood by one with ordinary skill in the art. The specific exampleswhich follow illustrate the methods in which the bioadhesive sealantcompositions of the present invention may be prepared in a clinicalsetting and are not to be construed as limiting the invention in sphereor scope. The methods may be adapted to variation in order to producecompositions embraced by this invention but not specifically disclosed.Further, variations of the methods to produce the same compositions insomewhat different fashion will be evident to one skilled in the art.

EXAMPLES

[0287] The examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the inventionin any way.

Example 1 Preparation of Bioadhesive Sealant Composition Using PlateletRich Plasma and Serum

[0288] 6 cc's of platelet rich plasma are drawn into receiving chamber961 and 3 cc's per PRP or PPP are drawn into receiving chamber 957 whichfurther contains 0.33 cc's of 10% calcium chloride and glass wool.Clotting of the contents will occur in two to eight minutes in receivingchamber 957. The clot is then squeezed through optional filter 958 andthe serum, produced therefrom, is added to the platelet rich plasmacontained in receiving chamber 961 by either mixing or spraying the twocomponents. The platelet rich plasma and the serum will gel withinapproximately three minutes.

[0289] The application of the gel using the syringe-type devices 902 asdescribed above maybe less than desirable for may applications.Consequently, in an alternate embodiment the inactive blood componentand thrombin can be mixed and/or injected into a mold having a desiredgeometric shape. The mold may be constructed of a material having awettable surface, such as, but not limited to plastic. In particular,platelet gel of the present invention may be used to temporarily fill,cavities such as but not limited to holes left in the gum from toothextraction and/or holes left in tissue or bone as a result of injury orsurgical procedures. The present invention provides a simpler way ofintroducing platelet gel for specific uses, by providing that theplatelet gel be pre-shaped or molded into a beneficial shape prior tobeing inserted into a cavity. In the case of tooth extraction theplatelet gel may be shaped so as to achieve a basic conical shape. Othershapes such as, but not limited to rods, and rectangles are contemplatedby this invention. The ability to cause the gel to be more, or less,solid and thus malleable may be achieved during the activation sequenceof the gel formation.

[0290] The foregoing description is considered as illustrative only ofthe principles of the invention. Furthermore, since numerousmodifications and changes will readily occur to those skilled in theart, it is not desired to limit the invention to the exact constructionand processes shown as described above. Accordingly, all suitablemodifications and equivalents may be resorted to falling within thescope of the invention as defined by the claims which follow.

[0291] The foregoing description is considered as illustrative only ofthe principles of the invention. The words “comprise,” “comprising,”“include,” “including,” and “includes” when used in this specificationand in the following claims are intended to specify the presence of oneor more stated features, integers, components, or steps, but they do notpreclude the presence or addition of one or more other features,integers, components, steps, or groups thereof. Furthermore, since anumber of modifications and changes will readily will readily occur tothose skilled in the art, it is not desired to limit the invention tothe exact construction and process shown described above. Accordingly,all suitable modifications and equivalents may be resorted to fallingwithin the scope of the invention as defined by the claims which follow.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A centrifuge forseparating components contained in a fluid, comprising: a flexiblecentrifuge bag having a doughnut shaped configuration and a centralcore; a rotatable rotor comprising an annular base having a firstannular channel and an annular cover having a second annular channel forholding a flexible centrifuge bag therebetween, whereby when said baseand said cover are superimposed, said first and second channels definean annular interior chamber having an off-centered figure eightconfiguration, said cover further comprising one or more radially spacedapart concentric indicator lines for monitoring the separation of saidcomponents.
 2. The centrifuge of claim 1, wherein said base furthercomprises a first outer rim extending from the base top surface and afirst grooved column axially centered and extending from the base topsurface, wherein the height of said first rim is equal to the height ofsaid first column, said first rim and said first column defining saidfirst annular channel, and wherein said cover further comprises a secondouter rim extending from the cover bottom surface and a second groovedcolumn axially centered and extending from the cover bottom surface,wherein the height of said second rim is equal to the height of saidsecond column, said second rim and said second column defining saidsecond annular channel.
 3. The centrifuge of claim 1, wherein saidflexible bag comprises superimposed upper and lower flexible sheetshaving doughnut shaped configurations, inner perimeters defining acentral core of said centrifuge bag, and outer perimeters, wherein saidupper and lower sheets are completely sealed together at their outer andinner perimeters, said centrifuge bag further comprising an inlet tubeand at least one outlet tube, said tubes sandwiched between said upperand lower sheets and sealed therebetween and extending from the centralcore of said centrifuge bag, wherein the distal end of one of saidoutlet tubes is positioned relative to a predetermined indicator line.4. The centrifuge of claim 2, wherein when said centrifuge bag ispositioned between said cover and base and wherein said tubes are seatedin said grooves.
 5. The centrifuge of claim 1, wherein said componentsseparate at varying radial distances based on their densities.
 6. Thecentrifuge of claim 3, wherein said fluid is whole blood and saidcomponents comprise a red blood cell fraction, a white blood cellfraction, a platelet rich plasma fraction, and a platelet poor plasmafraction.
 7. The centrifuge of claim 6, wherein said cover comprisesfirst, second and third indicator lines having radial distances thatindicate the location of the components with respect to the distal endon said outlet tube.
 8. The centrifuge of claim 7, wherein the distalend of one of said outlet tubes is positioned relative to said secondindicator line.
 9. A method for separating components contained in afluid, comprising: a) providing a flexible centrifuge bag comprising aninlet tube and at least one outlet tube; b) providing a rotatable rotorcomprising an annular base and an annular cover for holding saidflexible centrifuge bag therebetween, said base having a first outer rimextending from the base top surface and a first column axially centeredand extending from the base top surface, said first rim and said firstcolumn defining a lower chamber, said cover having a second outer rimextending from the cover bottom surface and a second column axiallycentered and extending from the cover bottom surface, said second rimand said second column defining an upper chamber, said cover furthercomprising one or more spaced apart concentric indicator lines formonitoring the separation of said components, and wherein the distal endof one of said outlet tubes is positioned relative to a predeterminedindicator line; c) positioning said flexible bag in said base, whereinsaid base column extends through said bag central core; d) superimposingsaid cover over said base, whereby said lower and upper chambers definean interior chamber and wherein said centrifuge bag is rotatable withsaid rotor; e) rotating said rotor; f) introducing an aliquot of saidfluid to said rotating centrifuge bag via said inlet tube, whereby saidcomponents separate into fractions based on their densities at varyingradial distances from the central axis; and g) removing a predeterminedfraction via said outlet tube.
 10. The method of claim 9, wherein saidcentrifuge bag comprises superimposed upper and lower flexible sheetshaving doughnut shaped configurations, inner perimeters defining acentral core of said centrifuge bag, and outer perimeters, wherein saidupper and lower sheets are completely sealed together at their outer andinner perimeters and wherein said inlet and said outlet tubes aresandwiched between said upper and lower sheets and sealed therebetween,said tubes extending from the central core of said centrifuge bag 11.The method of claim 9, wherein said first column further comprises afirst groove extending the diameter of said first column, and saidsecond column further comprises a second groove extending the diameterof said second column, wherein when said bag is held between said coverand base said tubes are seated in said grooves.
 12. The method of claim9, wherein said components separate at varying radial distances based ontheir densities.
 13. The method of claim 10, wherein said fluid is wholeblood and said components comprise a red blood cell fraction, a whiteblood cell fraction, a platelet rich plasma fraction, and a plateletpoor plasma fraction.
 14. The method of claim 13, wherein said covercomprises first, second and third indicator lines having radialdistances that indicate the location of the components with respect tothe distal end on said outlet tube.
 15. The method of claim 14, whereinthe distal end of said outlet tube is positioned relative to said secondindicator line.